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What can we learn from studying
fungal microbiota?
Laurence Delhaes1,2
- Romain Dassonneville3
laurence.delhaes@pasteur-lille.fr
1
BDEEP – EA4547 (CIIL), Institut Pasteur de Lille, Université de Lille 2 – France
2
Département de Microbiologie, Service de Parasitologie-Mycologie, CHU de Lille - France.
3
PEGASE-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. Cell Host & Microbe
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 dogma
The 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 analysis
Identification 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 clinical significance of filamentous fungi positive sputum cultu
Liu et al. J Cyst Fibros. 2013 May
Lung mycobiota in CF: Results of the pilot study
Fungal 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:

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What can fungal microbiota reveal about lung health in CF patients

  • 1. What can we learn from studying fungal microbiota? Laurence Delhaes1,2 - Romain Dassonneville3 laurence.delhaes@pasteur-lille.fr 1 BDEEP – EA4547 (CIIL), Institut Pasteur de Lille, Université de Lille 2 – France 2 Département de Microbiologie, Service de Parasitologie-Mycologie, CHU de Lille - France. 3 PEGASE-GèneDiffusion, Institut Pasteur de Lille, Université de Lille 2 – France
  • 2. 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. Cell Host & Microbe 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
  • 3.  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
  • 4.  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 dogma The 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
  • 5. 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
  • 6.  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
  • 7. 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)
  • 8. 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
  • 9. 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 analysis Identification 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
  • 10. 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 clinical significance of filamentous fungi positive sputum cultu Liu et al. J Cyst Fibros. 2013 May
  • 11. Lung mycobiota in CF: Results of the pilot study Fungal 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
  • 12. ⇒ 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
  • 13. ⇒ 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
  • 14. ⇒ 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
  • 15. 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
  • 16. 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
  • 17. 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]
  • 18. 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)
  • 19. 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
  • 20.  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
  • 21. 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)
  • 22. 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:

Notes de l'éditeur

  1. Good morning everyone.   1st, I would like to thank ESCF committee, Mister Moss and Mac Elvaney for giving this opportunity to share with you our data.
  2. La recherche sur le microbiome du poumon est un domaine relativement nouveau pouvant conduire à modifier notre vision des maladies respiratoires [1]. Les poumons des sujets en bonne santé ont longtemps été considérés comme stériles sur la base de données obtenues par les méthodes classiques, principalement fondées sur la culture bactérienne. De fait, le grand projet américain initié par le National Institute of Health (NIH) sur le microbiome humain n’incluait pas, jusqu’à récemment, les poumons comme un site de recherche [2]. Cependant, des données récemment publiées, basées sur des méthodes indépendantes des cultures bactériennes, ont démontré que les poumons de sujets sains non fumeurs étaient habitées par une population bactérienne, peu abondante mais diversifiée [3]. La fonction respiratoire est l’un des plus grands enjeux de demain en matière desanté publique: - Les troubles… -la BPCO sera la 4ème -la prévalence de l’asthme est en augmentation dans Pays développés (25 millions d’américains atteints asthme) -la mucoviscidose, pathologie à laquelle nous nous intéressons plus particulièrement, est la maladie génétique la plus fréquente dans la population caucasienne. (1/3000 -4000 Nnés en France). L’épaississement du mucus observé dans la mucoviscidose, a comme conséquence directe une accumulation (un piégeage) des microorganismes (bactérie champ) au niv pulmonaire, ce qui entrave fortement la fonction respiratoire, est responsable d’infection/ sur-infections pulmonaires (notamment les infections à Pseudomonas aeruginosa) = cause majeure de mortalité dans la mucoviscidose Au même titre que les sols, les océans (et autres milieux), il existe un microbiote de l’organisme humain: le plus étudié et documenté étant le microbiote intestinal . environ 100 000 milliards, soit au moins deux fois plus que le nombre moyen de cellules de l&amp;apos;organisme Mais de la même façon, il existe un microbiote cutané, vaginal, et biensur pulmonaire A ce jour c’est essentiellement le microbiote bactérien qui est étudié Rationnel: Aspergillus scedos : 2 plus frequent champ chez la muco P jiroveci: portage très fréquent muco + BPCO - Portage Pj associé aux stades sévères (III, et IV) de BPCO: role dans la réponse inflammatoire ?; 2.5% d’API chez les patients atteints de BPCO avec une mortalité très élévée 70-95%: signifcation de la colonisation sensibilisation ?
  3. Regarding fungi,   They are also present in various ecosystems (but poorly studied or analysed) They are playing an important role in soil regeneration. Fungi/molds (especially ascomycetes such as Aspergillus) have (along with bacteria) a key role in most land-based ecosystems – they are important decomposers, breaking down organic substances. One million and a half represents the number of fungus species currently estimated for the entire world. But only 6% have been identified   Microbes et environnement: grand intérêt+++ Microorganismes: impliqués dans de nombreux processus globaux Microorganismes: Rôle fondamental en biotechnologie (ATB, fermentation, expression, biomatériaux) the Ascomycota are heterotrophic organisms that require organic compounds as energy sources ils ne peuvent pas, comme les plantes, synthétiser leur matière organique à partir du CO2 atmosphérique. Ils doivent donc puiser dans le milieu ambiant l’eau et les substances organiques et minérales nécessaires à leurs propres synthèses ; ils sont hétérotrophes (Kendrick 2001).
  4. La concentration dans un environnement intérieur sans contamination fongique est généralement en dessous de 103 spores/m3 d’air (OMS 2009). La fonction respiratoire est l’un des plus grands enjeux de demain en matière desanté publique: - Les troubles… -la BPCO sera la 4ème -la prévalence de l’asthme est en augmentation dans Pays développés (25 millions d’américains atteints asthme) -la mucoviscidose, pathologie à laquelle nous nous intéressons plus particulièrement, est la maladie génétique la plus fréquente dans la population caucasienne. (1/3000 -4000 Nnés en France). L’épaississement du mucus observé dans la mucoviscidose, a comme conséquence directe une accumulation (un piégeage) des microorganismes (bactérie champ) au niv pulmonaire, ce qui entrave fortement la fonction respiratoire, est responsable d’infection/ sur-infections pulmonaires (notamment les infections à Pseudomonas aeruginosa) = cause majeure de mortalité dans la mucoviscidose Au même titre que les sols, les océans (et autres milieux), il existe un microbiote de l’organisme humain: le plus étudié et documenté étant le microbiote intestinal . environ 100 000 milliards, soit au moins deux fois plus que le nombre moyen de cellules de l&amp;apos;organisme Mais de la même façon, il existe un microbiote cutané, vaginal, et biensur pulmonaire A ce jour c’est essentiellement le microbiote bactérien qui est étudié Rationnel: Aspergillus scedos : 2 plus frequent champ chez la muco P jiroveci: portage très fréquent muco + BPCO - Portage Pj associé aux stades sévères (III, et IV) de BPCO: role dans la réponse inflammatoire ?; 2.5% d’API chez les patients atteints de BPCO avec une mortalité très élévée 70-95%: signifcation de la colonisation sensibilisation ?
  5. La concentration dans un environnement intérieur sans contamination fongique est généralement en dessous de 103 spores/m3 d’air (OMS 2009). La fonction respiratoire est l’un des plus grands enjeux de demain en matière desanté publique: - Les troubles… -la BPCO sera la 4ème -la prévalence de l’asthme est en augmentation dans Pays développés (25 millions d’américains atteints asthme) -la mucoviscidose, pathologie à laquelle nous nous intéressons plus particulièrement, est la maladie génétique la plus fréquente dans la population caucasienne. (1/3000 -4000 Nnés en France). L’épaississement du mucus observé dans la mucoviscidose, a comme conséquence directe une accumulation (un piégeage) des microorganismes (bactérie champ) au niv pulmonaire, ce qui entrave fortement la fonction respiratoire, est responsable d’infection/ sur-infections pulmonaires (notamment les infections à Pseudomonas aeruginosa) = cause majeure de mortalité dans la mucoviscidose Au même titre que les sols, les océans (et autres milieux), il existe un microbiote de l’organisme humain: le plus étudié et documenté étant le microbiote intestinal . environ 100 000 milliards, soit au moins deux fois plus que le nombre moyen de cellules de l&amp;apos;organisme Mais de la même façon, il existe un microbiote cutané, vaginal, et biensur pulmonaire A ce jour c’est essentiellement le microbiote bactérien qui est étudié Rationnel: Aspergillus scedos : 2 plus frequent champ chez la muco P jiroveci: portage très fréquent muco + BPCO - Portage Pj associé aux stades sévères (III, et IV) de BPCO: role dans la réponse inflammatoire ?; 2.5% d’API chez les patients atteints de BPCO avec une mortalité très élévée 70-95%: signifcation de la colonisation sensibilisation ?
  6. La concentration dans un environnement intérieur sans contamination fongique est généralement en dessous de 103 spores/m3 d’air (OMS 2009). La fonction respiratoire est l’un des plus grands enjeux de demain en matière desanté publique: - Les troubles… -la BPCO sera la 4ème -la prévalence de l’asthme est en augmentation dans Pays développés (25 millions d’américains atteints asthme) -la mucoviscidose, pathologie à laquelle nous nous intéressons plus particulièrement, est la maladie génétique la plus fréquente dans la population caucasienne. (1/3000 -4000 Nnés en France). L’épaississement du mucus observé dans la mucoviscidose, a comme conséquence directe une accumulation (un piégeage) des microorganismes (bactérie champ) au niv pulmonaire, ce qui entrave fortement la fonction respiratoire, est responsable d’infection/ sur-infections pulmonaires (notamment les infections à Pseudomonas aeruginosa) = cause majeure de mortalité dans la mucoviscidose Au même titre que les sols, les océans (et autres milieux), il existe un microbiote de l’organisme humain: le plus étudié et documenté étant le microbiote intestinal . environ 100 000 milliards, soit au moins deux fois plus que le nombre moyen de cellules de l&amp;apos;organisme Mais de la même façon, il existe un microbiote cutané, vaginal, et biensur pulmonaire A ce jour c’est essentiellement le microbiote bactérien qui est étudié Rationnel: Aspergillus scedos : 2 plus frequent champ chez la muco P jiroveci: portage très fréquent muco + BPCO - Portage Pj associé aux stades sévères (III, et IV) de BPCO: role dans la réponse inflammatoire ?; 2.5% d’API chez les patients atteints de BPCO avec une mortalité très élévée 70-95%: signifcation de la colonisation sensibilisation ?
  7. Nous nous sommes attachés à déterminer la composition du microbiote fongique du patient atteint de muco, en prenant également en compte les bactéries, Avec comme questions biologiques sous-jacentes (aux quelles nous avons essayé de répondre) : -Le microbiote du patient atteint de muco est-il différent de celui du patient sain? -Quelle est sa stabilité dans le temps? -Est-il corrélé à l’état clinique/évolution du patient? Et nous avons utilisé les outils qui nous semblaient les plus adaptés au contexte càd les approches DE SEQUENCAGE HAUT-DEBIT
  8. Notre stratégie méthodologique était basée sur le pyroséq sur automate 454 (roche) Je vais en donner les grandes étapes de cette méthodologie Si vous souhaitez nous pourrons en reparler après. -aà partir des expecto (8 de 4 patients adultes stables cliniquement), nous avons extrait l’AND total OU métagenome (kit Hight pure Kit – Roche) -2 PCR ciblant l’ADN16 (V3) des bactéries et le locus ITS2 des champignons ont été réalisées (grâce à 2 couples d’amorces) -Séquençage multi-parallélisé, obtention de plusieurs milliers de pyroséquences Elles ont été identifiées par comparaisosn à 2 banques de données la banque SILVA en accès libre pour les seq de 16S et une banque spécifique des séquences ITS2 que nous avons créé et validé pour cette étude en collaboration avec Genoscreen à IPL Après plusieurs étapes de bioinformatique, les pyroséquences sont groupées en fonction de leur identité puis attribuées à une espèce quand c’est possible ou sinon à un genre conformément aux données de nos 2 banques et selon les critères choisis. Puis nous avons réalisé une analyse phylogénétique des différents taxons permettant de comparer les 2 échantillons d’un même patient = grâce au logiciel MEGAN,
  9. - Pseudomonas reads were highly similar to P. aeruginosa strains isolated from CF patients or endotracheal tube biofilms. Notre stratégie méthodologique était basée sur le pyroséq Je vais en donner les grandes étapes de cette méthodologie Si vous souhaitez nous pourrons en reparler après. -a partir des expecto, nous avons extrait l’AND total -2 PCR ciblant l’ADN16 des bacteries et le locus ITS2 des champignons ont été réalisées (grace à 2 couples d’amorces) -Plusieurs milliers de pyroséquences ont été obtenus, Elles ont été identifiées par comparaisosn à 2 banques de données la banque SILVA en accès libre pour les seq de 16S et une banque specifique des séquences ITS2 que nous avons créé et validé pour cette étude en collaboration avec Genoscreen à IPL Après plusieurs étapes de bioinformatique, les pyroséquences sont groupées en fonction de leur identité puis attibuées à une espèce quand c’est possible ou sinon à un genre conformément aux données de nos 2 banques et selon les critères choisis. Puis nous avons réalisé une analyse phylogénétique des differents taxons permettant de comparer les 2 échantillons d’un même patient,
  10. At the physiology level, 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. fumigatu - All are known to be able to form biofilm consortia, and to produce direct and indirect microbe-microbe interactions including quorum-sensing phenomenon Establishing microbiota in CF airways = dynamic event managed (we can supppose) to be beneficial to all members of the microbial population, probably with some “synergene” phenomenon
  11. At the physiology level, 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. fumigatu - All are known to be able to form biofilm consortia, and to produce direct and indirect microbe-microbe interactions including quorum-sensing phenomenon Establishing microbiota in CF airways = dynamic event managed (we can supppose) to be beneficial to all members of the microbial population, probably with some “synergene” phenomenon
  12. Principal component analysis (PCA) is a mathematical procedure that uses an orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components. The number of principal components is less than or equal to the number of original variables. This transformation is defined in such a way that the first principal component has the largest possible variance (that is, accounts for as much of the variability in the data as possible), and each succeeding component in turn has the highest variance possible under the constraint that it be orthogonal to (i.e., uncorrelated with) the preceding components. Principal components are guaranteed to be independent only if the data set is jointly normally distributed. PCA is sensitive to the relative scaling of the original variables.
  13. The Addition of the 2 axes represents the explained part of the variance Principal component analysis (PCA) is a mathematical procedure that uses an orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components. The number of principal components is less than or equal to the number of original variables. This transformation is defined in such a way that the first principal component has the largest possible variance (that is, accounts for as much of the variability in the data as possible), and each succeeding component in turn has the highest variance possible under the constraint that it be orthogonal to (i.e., uncorrelated with) the preceding components. Principal components are guaranteed to be independent only if the data set is jointly normally distributed. PCA is sensitive to the relative scaling of the original variables.
  14. The Addition of the 2 axes represents the explained part of the variance Principal component analysis (PCA) is a mathematical procedure that uses an orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components. The number of principal components is less than or equal to the number of original variables. This transformation is defined in such a way that the first principal component has the largest possible variance (that is, accounts for as much of the variability in the data as possible), and each succeeding component in turn has the highest variance possible under the constraint that it be orthogonal to (i.e., uncorrelated with) the preceding components. Principal components are guaranteed to be independent only if the data set is jointly normally distributed. PCA is sensitive to the relative scaling of the original variables.
  15. Kappamyces is a new genus for a chytrid member of the Rhizophydium clade is described zoospore in the Chytridiales Granulicatella species, along with the genus Abiotrophia, were originally known as ‘nutritionally variant streptococci’. They are a normal component of the oral flora, but have been associated with a variety of invasive infections in man and are most noted as a cause of bacterial endocarditis. It is often advised that Granulicatella endocarditis should be treated in the same way as enterococcal endocarditis. Atopobium= Anearobie bacteria, involved in vaginosis. This inability to prevent recurrences reflects our lack of knowledge on the origins of BV. Atopobium vaginae has been recently reported to be associated with BV Gemella bacteria are primarily found in the mucous membranes of humans and other animals, particularly in the oral cavity and upper digestive tract.
  16. Definition of NECTRIACEAE. : a family of ascomycetous fungi (order Hypocreales) that are close to fusarium and currently environmental /phytopathogen Capnocytophaga is a genus of Gram-negative bacteria. Normally found in the oropharyngeal tract of mammals, they are involved in the pathogenesis of some animal bite wounds as well as periodontal diseases.[1]
  17. Definition of NECTRIACEAE. : a family of ascomycetous fungi (order Hypocreales) that are close to fusarium and currently environmental /phytopathogen Capnocytophaga is a genus of Gram-negative bacteria. Normally found in the oropharyngeal tract of mammals, they are involved in the pathogenesis of some animal bite wounds as well as periodontal diseases.[1]
  18. Definition of NECTRIACEAE. : a family of ascomycetous fungi (order Hypocreales) that are close to fusarium and currently environmental /phytopathogen Capnocytophaga is a genus of Gram-negative bacteria. Normally found in the oropharyngeal tract of mammals, they are involved in the pathogenesis of some animal bite wounds as well as periodontal diseases.[1]
  19. OW: raisonnable mais pas parfait (tobramycine n’impacterait pas la diversité) Pour conclure, cette étude est modeste : 8 echantillons mais à mon sens très prometteuse elle a clairement mis en évidence une correlation entre la diversité du microbiote et le statut du patient en utilisant le séquençage haut-débit et les outils de bioinfo correspondant(elle vient d’être publiée) Sur le plan physiopathologique, dans la mucoviscidose, la composition du mucus représente des conditions favorables au développement des co-infection / co-colinisation Il a été montré que La réduction de tension en oxygène (observée dans la muco) favorise la croissance de … De plus tous ces microorganismes sont capables de former des biofilms avec de potentielles interactions intra mais aussi inter-spécifiques La formation du microbiote est donc un évement dynamique, pour mieux le comprendre, il nous faut maintenant
  20. (1/3000 Nnés en France) (1/3000 Nnés en France) Rationel Aspergillus scedos : 2 plus frequent champ chez la muco P jiroveci: portage très fréquent muco + BPCO 2.5% d’API chez les patients atteints de BPCO avec une mortalité très élévée 70-95%: siginifcation de la colonisation sensibilisation ? Portage Pj associé aux stades sévères (III, et IV) de BPCO: role dans la réponse inflammatoire ?