What can we learn from studying fungal microbiota?

Laurence Delhaes1,2 - Romain Dassonneville3

laurence.delhaes@pasteur-lille.fr

1BDEEP – EA4547 (CIIL), Institut Pasteur de Lille, Université de Lille 2 – France2Département de Microbiologie, Service de Parasitologie-Mycologie, CHU de Lille - France.3PEGASE-GèneDiffusion, Institut Pasteur de Lille, Université de Lille 2 – France

Recently, lungs have been included in the Human Microbiome NIH project as a site of microbiota analysis. But bacterial microbiota analysis have been largely assessed while virome and mycobiome are significant/essential

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

Variations of the bacterial concentrations between sites: While trillions of commensal bacteria termed “the microbiota” are in close proximity to a single layer of epithelial cells in the colon [Arthur JC, et

al. . Science. 2012], only 20 to 1 bacteria per 1 000 human cells seem to evolve in lung tissue [Sze MA, et al. 2012; Charlson ES et al. 2011; Erb-Downward JR, et al. 2011]

Introduction: Human superorganism and Lung

Species number (bacteria)

Acid mine See Termite hindgut Human gut Soil

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

Playing an important role within soil regeneration (nutriment - metabolism of plant decomposition)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

Respiratory function: A major issue for Public Health

In relation with the outdoor environment As for the other air-breathing animals, human lungs are dealing with gas exchange (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 [WHO 2009]

Lungs: Sterile organs: an old dogmaThe presence of mucosal microbiomes appears to be the rule rather than

the exception[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)-Asthma 10-15% of the whole population in Europe & North America-Cystic Fibrosis (CF): Most common serious hereditary disorder in the Caucasian population [Rabe et al. « The year of the lung ». Lancet 2010]

Introduction: Fungal diversity and human 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 EID

[Fisher et al. Review in Nature 2012] Emerging fungal threats to animal, plant & ecosystem health

Medically important bacterial-fungal interactions

Introduction: Fungal diversity and EID

[Peileg et al. www.Nature.com/reviews/micro2010]

Human diseases can develop from an imbalance between commensal bacteria and fungi or from invasion of particular host niches by opportunistic bacterial and fungal pathogens.

Bacteria and fungi directly and indirectly influence each other in several ways

Introduction: Fungal diversity and EID

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)

Purpose: 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?

Introduction: Fungal diversity and human Lung

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: Metagenomic approach

Lung mycobiota in CF: Results and Discussion

2 studies: 1) A pilot study to validate our approach

2) An ongoing study to analyze the relevance of fungi in CF pulmonary exacerbation→ with the idea of deciphering the place of Aspergillus spp.

→ the role of ABPA (allergic bronchopulmonary aspergillosis) in such exacerbation?

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 cl inical signif icance of f i lamentous fungi posit ive sputum cultures in patients with cystic f ibrosis? Liu et al. J Cyst Fibros. 2013 May

Lung mycobiota in CF: Results of the pilot studyFungal diversity

involved in respiratory or infectious diseases

- The median [IQ] number of microorganism genera per sputum sample was 3.5 [3; 7.5] micromycetes [Bouchara et al. 2009].

- Among the 24 species /genera identified as fungi using deep-sequencing, only 4 have been isolated by cultures.

- This genomic method allowed the identification of additional species that are recognized as microorganisms

⇒ Validation of the molecular approach (ITS2 DB+++)

⇒ We observed a decrease of diversity and richness for fungal and bacterial communities significantly associated with poor clinical status (S-K score and BMI) and decreased lung function (FEV1 and FVC)

Lung mycobiota in CF: Results of the pilot study

⇒ Mucus composition in CF provides conditions suitable for chronic co-infection: - Reduced oxygen tension in CF lung favourable for growth of P. aeruginosa, anaerobes (i.e. SMG members), C. albicans, and A. fumigatus

- All are known to be able to form biofilm consortia, and to produce direct and indirect microbe-microbe interactions including quorum-sensing phenomenon

[Rybtke et al. 2011; Bandara et al. 2010; Mowat et al. 010; Sibley et al. 2008; Alvarez-Ortega et al. 2007; Dimitru et al. 2007; Semighini et al. 2006]

Lung mycobiota in CF: Results of the pilot study

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

⇒ Microbial analysis done: (i) using deep-sequencing fungal/bacterial analysis (ii) using RT-PCR targeting RNA respiratory viruses (Seeplex RV15 ACE Detection kit (Seegene))

⇒ Statistical approach under process a first PCA (principal component analysis) taking into account the whole set of variables (40 per patient) for analyzing mycobiota versus 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).

Lung mycobiota: Relevance in CF exacerbation

Lung mycobiota in CF: Relevance 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

Lung mycobiota in CF: Relevance 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

Lung mycobiota in CF: Relevance 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]

Lung mycobiota in CF: Relevance in CF exacerbation

Aspergillus - Unfortunately, our PCA model

doesn’t explained this mold

- 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)

Lung mycobiota in CF: Relevance in CF exacerbation

Malassezia - As some anaerobes, M. furfur and

M. sympodialis are difficult to culture, both obligatory lipophilic. = skin flora yeasts of humans

Classically, they are associated with superficial infections of the skin (pityriasis versicolor - folliculitis)

They appear + correlated with anaerobes in agreement with their lipophyly (since anarobes can produce fatty acids)

ChromAgar Malassezia

Diversity indexes (Simpson and Shannon’s indexes) of bacteria and fungi appear equivalent

They seem to be independent variables (with a tendency of a positive correlation estimated at 0.18 & 0.3 for Shannon & Simpson’s indexes, respectively) → Integrate ARN-virus data→ Continue statistical analysis focusing on streptococcus species and less abundant (but more diverse) components of the mycobiota (rare biosphere - <0.1%)

Lung mycobiota in CF: Relevance in CF exacerbation

Lung mycobiota in CF: Concluding remarks

→ Candida & A. fumigatus = Main species/genus isolated [Charlson et al. 2012; Delhaes et al. 2012]

→ Role in the decrease of pulmonary function

→ Which place for fungi in CF exacerbation? Larger studies are now required to better understand CF

exacerbation associated communities (characterizing “attack & climax" metagenomes / metatranscritptomes)

→ What is mycobiota evolution and role when patients are treated with ATB cures? [Muco-Bac-Myco project - F Botterel & L Delhaes under process]

Comparing fungal and bacterial microbiota before and after a systemic antimicrobial treatment and correlating its evolution with the clinical outcome

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

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: