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Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq Tutor : Jacqueline Bert

BUDGET & TIMING

EXPERIMENTAL APPROACH

CONTEXT

  • Aspergillus fumigatus can cause Aspergillosis infection to immunocompromised patients.
  • Toxic treatments are already used: Fluconazole and Amphotericin B
  • We proposed an alternative with essential oils
  • Determine the optimal concentration on A.nidulans
  • Screening of EOs on A.nidulans
  • Study of synergistic effect of EOs on both fungi
  • Depending on those results, EOs' fungistatic effect will be tested against fungicide effect.
  • Toxicity on human cells will be assessed thanks to viability ratio

1st experimental week: Focus on synergistic effect of EOs mixes2nd experimental week: Focus on the cell culture and the fungistatic state of EOs

Need 827,76€ :-> Grant of 600€ + current budget of 200€

EXPECTEDOUTCOME

  • Highly effective=
    • Thyme, Oregano, Cinnamon
  • Optimal mixes=
    • Tea tree/Oregano
    • Peppermint/Oregano,
    • Cinnamon/Oregano or Thyme

AIMS

To find an alternative treatment to Aspergillosis

  • Select effective essential oils
  • Determine the optimal mix(es) of EOs
  • Test if fungistatic can become fungicide
  • Perform toxicity tests

REFS.

ABSTRACT

COLLAB.

Sup'Biotech Interactive Scientific Poster ©

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

CONTEXT

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

  • Aspergillosis is a disease caused by Aspergillus fumigatus
  • Present everywhere in the external environment
  • Is an opportunistic and filamentous fungus
  • Disseminates easily

Morphology of Aspergillus fumigatus

[1] LATGE J.P., “Aspergillus fumigatus and aspergillosis”, Clinical Microbiology Reviews, vol 12, n°2, April 1999, pp 310-350 [2] SUGUI J.A., KWON-CHUNG K.J. et al, “Aspergillus fumigatus and Related Species”, Cold Spring Harbor Perspectives in Medicine, vol 5, n°2, Februrary 2015 [3] NC, « Aspergillose », Institut Pasteur, launched on : January 2013, available on : https://www.pasteur.fr/fr/centre-medical/fiches-maladies/aspergillose

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

CONTEXT

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

  • Aspergillosis is responsible of 90% of immunocompromised patients' infections
  • Causes invasion of fungi in the respiratory tract
  • It is currently treated with two chemicals compounds : Amphotericin B and Fluconazole

Infection of the immunocompromied patient with A.fumigatus

[1] LATGE J.P., “Aspergillus fumigatus and aspergillosis”, Clinical Microbiology Reviews, vol 12, n°2, April 1999, pp 310-350 [2] SUGUI J.A., KWON-CHUNG K.J. et al, “Aspergillus fumigatus and Related Species”, Cold Spring Harbor Perspectives in Medicine, vol 5, n°2, Februrary 2015 .

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

CONTEXT

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

  • Amphotericin B and Fluconazole induce serious adverse effects.
  • One solution: use alternative treatments, such as essential oils (EOs) with antifungal properties.
  • Use of Aspergillus nidulans and Aspergillus oryzae as models
  • This solution has already been considered in a previous study at Sup'Biotech: "Antifungal properties of Essential Oils against Aspergillosis"
    • On A.oryzae were tested antifungal properties of various EOs
    • Thyme Thymol and Oregano compact EOs have the most effective antifungal activities

[4] BOUTTE Dauphine, CALMET Julie et al, “Antifungal properties of Essential Oils against Aspergillosis”, Journal of Sup’Biotech, January 2020

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

AIMS

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

=> Find an alternative treatment to aspergillosis by using essential oils To accomplish our objective, the following experiments will be done:

  • Select effective essential oils against A. nidulans growth.
  • Determine optimal mix(es) of essential oils having fungicide effects on A.oryzae and A.nidulans.
  • Test if fungistatic effect becomes fungicide according to the concentration of EOs and the time.
  • Perform toxicity tests using human pulmonary cells.

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

EXPERIMENTAL APPROACH

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

First, optimal concentration of A. nidulans will be determined to easily follow the antifungal effect.

[4] BOUTTE Dauphine, CALMET Julie et al, “Antifungal properties of Essential Oils against Aspergillosis”, Journal of Sup’Biotech, January 2020

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

EXPERIMENTAL APPROACH

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

Mixes of 3 EOs will be test by the same principle.

[4] BOUTTE Dauphine, CALMET Julie et al, “Antifungal properties of Essential Oils against Aspergillosis”, Journal of Sup’Biotech, January 2020

EXPERIMENTAL APPROACH

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

[5] POIROT Tony, Bon usage des huiles essentielles, effets indésirables et toxicologie, thèse de pharmacie, under the direction of Mr Luc FERRARI, University of Lorraine, 2016, page : 87 [6] Pr BAEZA-SQUIBAN Armelle, « Méthodes d’évaluation de la toxicité. Toxicologie expérimentale in vitro et in vivo », Laboratoire des réponses moléculaires et cellulaires aux xénobiotiques, University Paris-Diderot, available on : http://www.centres-antipoison.net/paris/DIU_Tox_Med_2017_2018/20180112/DIU_Tox_Med_2017_18_A_Baeza_Squiban_Evaluation_tox_vivo_vitro.pdf

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

TIMING

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

Gantt Chart

1st week of experimentation:

  • Test of EOs mixes on A. oryzae
  • Test of single EOs on A. nidulans
  • Preparation of cells

2nd week of experimentation:

  • Fungistatic test on A. oryzae and A. nidulans
  • Toxicity test of EOs on cancerous cells

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

BUDGET

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

Need 827,76€

Current budget: 200€ Ask for Grant subvention: 600€

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

EXPECTED OUTCOME

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

  • Fungistatic -> fungicide: Peppermint, Lavender and Tea tree have a fungistatic effect which can become fungicide.

inhibition

Positive control (Amphotericin B)

Negative control

(control)

  • Cellular toxicity: we expect EOs and mixes to be toxic at concentrations above 1 mg/mL

[4] BOUTTE Dauphine, CALMET Julie et al, “Antifungal properties of Essential Oils against Aspergillosis”, Journal of Sup’Biotech, January 2020

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

REFERENCES

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

[1] LATGE J.P., “Aspergillus fumigatus and aspergillosis”, Clinical Microbiology Reviews, vol 12, n°2, April 1999, pp 310-350 [2] SUGUI J.A., KWON-CHUNG K.J. et al, “Aspergillus fumigatus and Related Species”, Cold Spring Harbor Perspectives in Medicine, vol 5, n°2, Februrary 2015 [3] NC, « Aspergillose », Institut Pasteur, launched on : January 2013, available on : https://www.pasteur.fr/fr/centre-medical/fiches-maladies/aspergillose [4] BOUTTE Dauphine, CALMET Julie et al, “Antifungal properties of Essential Oils against Aspergillosis”, Journal of Sup’Biotech, January 2020 [5] POIROT Tony, Bon usage des huiles essentielles, effets indésirables et toxicologie, thèse de pharmacie, under the direction of Mr Luc FERRARI, University of Lorraine, 2016, page : 87 [6] Pr BAEZA-SQUIBAN Armelle, « Méthodes d’évaluation de la toxicité. Toxicologie expérimentale in vitro et in vivo », Laboratoire des réponses moléculaires et cellulaires aux xénobiotiques, University Paris-Diderot, available on : http://www.centres-antipoison.net/paris/DIU_Tox_Med_2017_2018/20180112/DIU_Tox_Med_2017_18_A_Baeza_Squiban_Evaluation_tox_vivo_vitro.pdf

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus oryzae against Aspergillosis

ABSTRACT

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

Aspergillosis is a disease caused by the fungus Aspergillus fumigatus that affects immunocompromised persons. It is currently treated with the two chemical compounds Amphotericin B and Fluconazole, which are toxic for kidneys. This study aims at finding an alternative treatment to Aspergillosis by using essential oils, known for their therapeutic properties. Mixes of two/three essential oils with different concentrations will be tested against ​A. oryzae and A. nidulans, two non-pathogenic models close to A. fumigatus.

COLLABORATIONS & ACKNOWLEDGEMENTS

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus Oryzae against Aspergillosis

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

  • Collaborations:
    • Sup'Biotech Researchers tutors (Mrs Bert, Mrs Mogensen and Mr Yates), class 1 microbiology laboratory
    • LRPIA laboratory Mrs Saint-Pol, provides both fungi species used
    • CEA laboratory Mrs Perdiz, provides pulmonary cancerous cell line
  • Potential collaboration : Pasteur Institute Jean-Paul Latgé, director of the Aspergillus unit. Class 2 microbiology laboratory to test on A.fumigatus
Our group acknowledges Jacqueline Bert to have allowed us to realise this project and also for her great help. We acknowledge Estelle Mogensen for her really useful advices and help in our project, Vanessa Perdiz and Agnès Saint-Pol for providing us cells and fungi as well as for their help concerning cultures and their advices. We also acknowledge Franck Yates for his advices and guidance during the project.

COLLABORATIONS & ACKNOWLEDGEMENTS

Study of therapeutic effect of essential oils on Aspergillus nidulans and Aspergillus Oryzae against Aspergillosis

Marylou Bouriot, Alexine Chemin, Isciane Commenge, Floricia Crusset, Manon Leclercq

  • Collaborations:
    • Sup'Biotech Researchers tutors (Mrs Bert, Mrs Mogensen and Mr Yates), class 1 microbiology laboratory
    • LRPIA laboratory Mrs Saint-Pol, provides both fungi species used
    • CEA laboratory Mrs Perdiz, provides pulmonary cancerous cell line
  • Potential collaboration : Pasteur Institute Jean-Paul Latgé, director of the Aspergillus unit. Class 2 microbiology laboratory to test on A.fumigatus
Our group acknowledges Jacqueline Bert to have allowed us to realise this project and also for her great help. We acknowledge Estelle Mogensen for her really useful advices and help in our project, Vanessa Perdiz and Agnès Saint-Pol for providing us cells and fungi as well as for their help concerning cultures and their advices. We also acknowledge Franck Yates for his advices and guidance during the project.