Mold and cystic fibrosis : what can we learn from studying fungal microbiota ?

Mold and Cystic fibrosis: What can we learn from studying fungal microbiota?

Laurence Delhaes1,2, Sebastien Monchy3, Magali Chabé1, Anne Prévotat2, Benoit Wallaert2, Eric Viscogliosi1, Christophe Audebert 4, Romain Dassonneville4

[email protected]

1BDEEP – EA4547 (CIIL), Institut Pasteur de Lille, Université de Lille 2 – North of France2Lille Hospital, CHU de Lille – North of France3Université Littoral Côte d’Opale, Boulogne – North of France4PEGASE-GèneDiffusion, Institut Pasteur de Lille,– North of France

Indo-French Seminar/workshop, 20-22nd January 2014)

Microorganisms (bacteria, archaea, yeasts, moulds, viruses) are colonizing all ecological systems

Such microorganisms are present even in extreme environments

A majority of these microorganisms remains to be identified

1-4 106 bacteria / g of soil (tropical rain forest)

2 108 cells / g of soil(desert)

1,04 1020 cells / cm3 of water(hypersaline water)

Introduction Worldwide microbial diversity

Micromycetes: are present in various ecosystems (but poorly studied/analyzed)

Playing an important role within soil regeneration (Heterotrophism)Of note: Fungi (especially ascomycetes) have/fulfill along with bacteria a central role in most land-based ecosystems, as they are important decomposers, breaking down organic substances.

1 500 000 represents the number of fungus species estimated for the entire earth/world But only 97 000 have been identified[Hibbett et al. 2007, Mycol Res , 111: 509-547]

Introduction Microbial diversity: Place of the fungi

As other ecological systems, there is a microbial diversity of human organisms

Introduction Human Microbial diversity

Species number (bacteria)

Acid mine See Termite hindgut Human gut Soil

2008: (i) European project MetaHIT, and the(ii) American “Common Fund's Human

Microbiome Project (HMP)” have been developed

→ to characterize the microbial communities found at different sites on the human body,

→ to analyze and compare the role of these microbes in human health and disease.

Proctor LM (2011) The Human Microbiome Project in 2011 and Beyond. Cell Host & Microbe 10:287-91

The main bacteria isolated in Humans are belonging 4 phyla (among the 50 known phyla). There are Firmicutes (in blue), Bacteroidetes (in pink), Actinobacteria (in green), and Proteobacteria (in purple). Body sites: nasal passages, oral cavities, skin, gastrointestinal tract, and urogenital tract

http://www.larecherche.fr/content/recherche/article?id=25319

Human beings: Which bacteria are living in us (The genomes in our genome)?

[La Recherche – a 2011 up-date: 1st panorama drawn from 7 studies realised from 2004 to 2007]

Introduction Human Microbial diversity

2 Missing elements

lungs = included in NIH project as a site of microbiota analysis. But bacterial microbiota analysis have been mainly assessed while virome and mycobiome are significant / essential

Respiratory function: A major issue for Public Heath

In relation with the outdoor environment Every day we breath 20,000 times, thereby inhaling ~10,000 liters of air (drawing and expulsion of air; 15m3 of air / day / adult) with a fungal contamination from to 108 to 103 spores/m3 in working to domestic usual exposure [OMS 2009; Pashley 2012]

Lungs: Sterile organs? [Morris et al. 2013; Beck et al.

2012; Erb-Downward et al. 2011; Huang et al. 2011]

-Respiratory disorders: 1st cause of worldwide consultations-Chronic obstructive pulmonary disease (COPD): 4th origin in worldwide decease by 2030 (WHO)-Cystic Fibrosis (CF): Most common serious hereditary disorder in the Caucasian population [Rabe et al. « The year of the lung ». Lancet 2010]

Introduction Human Microbial diversity and Lung

Lung microbial diversity in Cystic Fibrosis (CF):

- Lung diversity = Bacterial microbiota exists in healthy people [Morris et al. 2013; Beck et al. 2012; Erb-Downward et al. 2011; Huang et al. 2011]

- This bacterial community has been largely studied in CF, and seems to be associated with the evolution of the respiratory function in CF [Maughan et al. 2012; Guss et al. 2011; van der Gast et al. 2011; Rogers et al. 2010; Armougom et al. 2009; Bittar et al. 2008; Sibley et al. 2008; Tunney et al. 2008; Harris et al. 2007Goddard et al. 2012; Madan et al. 2012; Fodor et al. 2012]

Introduction Human Microbial diversity and Lung

The emerging world of the fungal microbiome

[Huffnagle et al. Trends in Microbiology 2013]

Nobody is fungus-free

Human fungal microbiome is part of the rare biosphere of the entire digestive microbiome

Evaluated at less than 0.1% of the genus in fecal material (from the MetaHIT group analysis)

Introduction Human Microbial diversity and Lung

Authors argue that human activity is intensifying fungal disease dispersal by modifying natural environments and thus creating new opportunities for evolution

Introduction Fungal diversity and Emerging Infectious Diseases

[Fisher et al. Review in Nature 2012]

Pathogenic fungi are emerging as major threats to animal, plant & ecosystem health

[From D. Denning, 2010]

Patterns of Aspergillus interactions with humansillustrating different host pathogen interactions, based on the

host damage response framework

Introduction Clinical features of Aspergillosis

Invasive Aspergillosis +/- disseminated

Local infection (aspergilloma, sinusitis)

Hypersensitivity disease (ABPA or Hinson- Pepys disease)

Pulmonary Mycosis

Immuno-allergy Mycosis

Invasive Aspergillosis = Poor Prognostic (mortality about 60-80%) → Difficult to diagnose, to treat

= Criteria of IA [De Pauw; Walsh 2008]

Air transmission Air transmission

Opportunist fungiOpportunist fungi

Fever unresponsive to broad–spectrum antibiotics with neutropenia (500/mm3, 10

days) Pulmonary symptoms early in the course, ± cutaneous or central nervous metastases

Patient at risk of IA: Immunocompromised patients

• OncoHaematology 3% – 14% >> allo-CT [Castagnola 2008; Leeflang 2008; Neofytos 2013]

• IA mortality of 56% in France [Lortholary 2011] vs 57.5% in US [TRANSNET, Kontoyannis 2010 ]

• Solid Transplantation : Pulmonary Transplantation IA = 4-6%, Major incidence in CF [Iversen 2008]

Non-neutropenic patients

• Corticotherapy [Lewis 2008; Castagnola 2008]

• Biotherapy (anti-TNF / Rituximab®) +++[Stankovic K, 2006; Lebeaux 2009; Delhaes 2010 ]

• Chronic pulmonary diseases (COPD) 2.5% [Guinea 2009; Ader 2006]

Introduction Clinical features of Aspergillosis

Computed tomography (CT) of the chest may be used to identify the halo sign (Fig1.) a macronodule surrounded by a perimeter of ground-glass opacity= an early but transitory sign of invasive pulmonary aspergillosis (IPA) (61% according to R. Greene et al.

2007)Macronodule is also a frequent but not specific sign (94% according to R. Greene et al. 2007)Later: air-crescent signs (Fig2.) (10% according to R. Greene et al. 2007). It corresponds to an excavation (corresponding to a central necrosis area when neutrophil cells are increasing and immune response is restored www.learningradiology.com

IPA= Fever unresponsive to broad–spectrum antibiotics with neutropenia and pulmonary symptoms early in the course

Introduction Lung mycobiota in CF : backgrounds

Immune hyperactivity to A. fumigatus chronic colonization

→ 2%, 5-15% in Asthma, CF patient population [Denning 2003; Blandin 2008; Gangneux 2008]

→ clinical diagnosis difficult (easier in case of pulmonary exacerbation)

→ Hyperthermia, Dyspnea, pulmonary infiltrates

Introduction Clinical features of Aspergillosis

Hyperthermia, Dyspnea, pulmonary infiltrates

Chest X-ray shows bilateral pulmonary infiltrates (right > left) [lungindia.com]

→ Not specific of ABPA

→ ABPA clinical features (as well as criteria) are usually overlapping the underlying disease, making the diagnosis uneasy

Introduction Clinical features of Aspergillosis

Immune hyperactivity to A. fumigatus chronic colonization

→ 2%, 5-15% in Asthma, CF patient population [Denning 2003; Blandin 2008; Gangneux 2008]

→ clinical diagnosis difficult (easier in case of pulmonary exacerbation)

→ Hyperthermia, Dyspnea, pulmonary infiltrates

→ Associated with immediate hyper-sensitivity (hypereosinophilia, serum total IgE) and IgG-mediated immune response

Introduction Clinical features of Aspergillosis

Patient serum

Ouchterlony method

Total Ag of A. fumigatus

Total Ag of A. flavus

4-5 precipitin bands (Ag-Ac complex) with high intensity

An ongoing study to analyze the relevance of fungi in CF pulmonary exacerbation

→ Pulmonary exacerbation = key event in CF lung alteration→ the role of ABPA (allergic bronchopulmonary aspergillosis)

in such exacerbation?→ with the idea of deciphering the place of Aspergillus spp

Aspergillus spp. especially A. fumigatus isolated from respiratory secretions is often a dilemma for the CF clinician in terms of clinical relevance and treatment

What is the clinical significance of filamentous fungi positive sputum cultures in patients with cystic fibrosis? Liu et al. J Cyst Fibros. 2013 May

Purpose Clinical features of Aspergillosis

Aim: What is the fungal microbiota (or Mycobiota) of CF patients?

Is the fungal microbiota stable?

Are the mycobiota diversity and richness associated to the clinical

status of CF patient? …

What is the fungal composition of lung microbiota in CF?

Mycobiota analysis by developping and using high throughput sequencing approach

Which relation we observed between the mycobiota and the bacterial composition?

Purpose Studying lung mycobiota in CF

DNA Extraction depends on matrix/substrate

PCRs targeted conserved genes that allow the amplification of species distant/different

phylogenetically (V3 of 16s rDNA – ITS2)

Massive sequencing (multi-parallelized, 454 FLX system) – getting hundreds of thousands of reads

Bio-informatic analysisIdentification by local blast to 2 databases: BLASTN ≠

- Silva SSU rRNA database release 102 - ITS2dbScreen that we designed de novo

Read assignments and clustering (at the species or genus level)

To allow a biologic analysis of the data, comparison between samples

(diversity analysis using MEGAN, U-clust, MEGANE5 progamms)

Collected sputum samples of CF patients

Materials & Methods Deep-sequencing approach

36 sputum samples From patients with (18) and without (18) pulmonary exacerbation were compared (clinical, radiological, biological data)

Microbial analysis done: (i) Microbial cultures

(ii) using RT-PCR targeting RNA respiratory viruses (Seeplex RV15 ACE Detection kit (Seegene))

(iii) using deep-sequencing fungal/bacterial diversity analysis

Statistical approach under process a first PCA (principal component analysis) taking into account the whole set of variables for analyzing mycobiota vs bacterial microbiota at the genus level we limited our analyses to the number of genera that were present at least in 3 patients and the number of OTU present at 1% (relative abundance).

Materials & Methods Lung Microbiota: Relevance in CF exacerbation

36 Sputum samplesA) S-K score= Score de Shwachman-KulczyckiWithout exacerbation With exacerbation

Results Lung Microbiota in CF exacerbation

A) S-K score= Score de Shwachman-KulczyckiWithout exacerbation With exacerbation

B) Rhinovirus

Other RN viruses

C) SMG

D) Mould

We confirme the significant association between HRV and pulmonary exacerbation[Goffard et al. 2014 in revision]

Conventional microbial analysis: (i) Microbial cultures(ii) RT-PCR targeting RNA respiratory viruses (Seeplex RV15 ACE Detection kit - Seegene)

36 Sputum samples

Results Lung Microbiota in CF exacerbation

No association between Milleri group streptococcus and pulmonary exacerbation

Only 2 patients colonized with Scedosporium apiospermum

No association between A. fumigatus and exacerbation

36 sputum samples From patients with (18) and without (18) pulmonary exacerbation were compared (clinical, radiological, biological data)

Microbial analysis done: (i) Microbial cultures

(ii) using RT-PCR targeting RNA respiratory viruses (Seeplex RV15 ACE Detection kit (Seegene))

(iii) using deep-sequencing fungal/bacterial diversity analysis

Statistical approach under process a first PCA (principal component analysis) taking into account the whole set of variables for analyzing mycobiota vs bacterial microbiota at the genus level we limited our analyses to the number of genera that were present at least in 3 patients and the number of OTU present at 1% (relative abundance).

953 999 reads size from 315 to 468 pb - 2/3 16s rDNA + 1/3 ITS2 Optimal rarefaction curves Modelization under process

Results Lung Microbiota in CF exacerbation

According to PCA graph:

Addition of the 2 axes = the explained part of the variability → 33% [42% in Zemanick et al. 2013]

For each variable, arrow lengh is proportional to the load of the corresponding variable on the first 2 principal components (Dim/axes 1-2) (the longer the arrow is = the more the axes explained the variable)

Our model and axes explained a lot of microorganisms

Results Lung Microbiota in CF exacerbation

Key point to read a PCA graph:

Interpreting a correlation between microorganisms as follow

Right angle =No correlation

Acute angle = Positive correlation

180° angle =Negative correlation

Results Lung Microbiota in CF exacerbation

Pseudomonas

- is alone [Zemanick et al. 2013]

- not correlated with “Malassezia plus Prevotella group” [Zemanick et al 2013]

- neither with the “Candida plus Rothia group” (which is not well explained by our axes since the arrows are short)

- but is negatively correlated with the “group of oral flora including streptococcus plus some environmental fungi”, as well as FEV1 – SK-score [Zemanick et al. 2013]

Results Lung Microbiota in CF exacerbation

Aspergillus - Unfortunately, our PCA model

explained poorly this mold (short arrows, anti-correlated to SK-score, FEV1, ),

- Neither exacerbation status: There was no differentiation between the group of patients with and without pulmonary exacerbation (according to PCA-barycenter of each patient group)

Results Lung Microbiota in CF exacerbation

**

→ Continue statistical analysis focusing on streptococcus species and less abundant (but more diverse) components of the mycobiota (rare biosphere - <0.1%)

Determining exhaustively the microbial community composition in CF patient sputa.

Developing new approaches based on deep-sequencing, (standardization, ARN analysis)

Improving management/survival of CF patients Development of ex vivo model biofilm to adapt drug

treatment (anti-bacterial/fungal) Predict the efficiency of drug treatment

Lung mycobiota

Improving our knowledge of microbiome by

Lung mycobiota in CF: Concluding remarks

Mycobiota = dynamic event, part of the overall lung microbiome (consisting of dynamic communities of virus, bacteria, & fungi)

Larger studies are now required to better understand associated fungal communities in CF, ABPA Studying mycobiota evolution when patients are treated

with ATB cures [Muco-Bac-Myco project - F Botterel & L Delhaes ongoing project]

International study to decipher lung microbiome evolution during exacerbation in CF patients [IMAGin-CF, submitted]

Institut Pasteur-Lille / Université de Lille 2• Laurence Delhaes • Eric Viscogliosi • Eduardo Dei-Cas• Anne Goffard• Magali Chabé

Université Littoral Côte d’Opale• Sébastien Monchy

• Christine Hubans / Stéphanie Ferreira

Faculté de Médecine de Lille• Benoit Wallaert• Anne Prévotat• Julia Salleron• Fréderic Wallet• Rodrigues Dessein• Sylvie Leroy

Société Genoscreen-Lille

Département de Microbiologie AP-HP Créteil •Françoise Botterel•Odile Cabaret•Jean-Winoc Decousser•Jean-Philippe Barnier

Consortium Pegase• Christophe Audebert / Romain Dassonneville

Multidisciplinary approaches (due to the massive data generated)

Collaborations: