FUNGAL SPORES Michel Thibaudon, Samuel Monnier, Julien Clertant In collaboration with Bernard CLOT and with slides from Dr. M. Muilenberg, Cambridge U., USA Dr. D. Job, U. of Neuchâtel, CH Inspired from « Mycology of the air » Michel Thibaudon – March 2018

Disclosure In relation to this presentation, I declare the following, real or perceived conflicts of interest: Type Company Employment full time / part time Resau National de Surveillance Aérobiologique Spouse / Family member employment / engagement None Research Grant (P.I., collaborator or consultant; pending and received grants) Other research support Speakers Bureau / Honoraria Ownership interest (stock, stock-options, patent or intellectual property) Consultant / advisory board A conflict of interest is any situation in which a speaker or immediate family members have interests, and those may cause a conflict with the current presentation. Conflicts of interest do not preclude the delivery of the talk, but should be explicitly declared. These may include financial interests (e.g. owning stocks of a related company, having received honoraria, consultancy fees), research interests (research support by grants or otherwise), organisational interests and gifts.

Michel Thibaudon – March 2018 Classification Michel Thibaudon – March 2018

Michel Thibaudon – March 2018

Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 Eukaryotes: 5 kingdoms MONERES PROTISTS PLANTS FUNGI ANIMALS Fungi: eukaryotes, heterotrophic, filamentous (hyphae  mycelium), cell wall made from chitin, sexual and asexual reproduction by spores. Michel Thibaudon – March 2018

Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 Fungi ~1.500.000 species, or more ~160.000 species described Saprophytic or parasitic Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 CLASSIFICATION Domain Kingdom Divisions Subdivisions Basidiomycetes Eumycètes Eucaryote Fungi or Mycetes Ascomycetes Myxomycètes ?? Deuteromycetes Zygomycetes Mastigomycetes Michel Thibaudon – March 2018

Fungal spores primary functions: to tide over unfavorable periods to disperse the fungus to new food sources (meiospores) genetic recombination Fungal spores take-off is often an active mechanism Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 Basidomycetes Basid  4 basidiospores meiospores, exospores never septate never branched bilateral symetry apiculus (attachment point) usually short (lenght / width ratio smaller than 4:1) sometimes a pore Michel Thibaudon – March 2018

Basid and basidiospores Michel Thibaudon – March 2018

Basid and basidiospores Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 Ascomycetes Ascus  8 ascospores meiospores, endospores septate or not groups of 8 (sometimes) often long (more than 4 times longer than wide) never birth scars never germination pores never branched Michel Thibaudon – March 2018

Michel Thibaudon – March 2018

Ascospores No attachment points; variable size, shape, septation B.Kendrick drawing Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 asci Michel Thibaudon – March 2018

(Deuteromycetes) = (Fungi imperfecti) = Mitosporic fungi (~moulds) mitospores = conidia (sing. conidium) (asexual) produced at the end of specialised hyphae always a central birth scar frequently septate can be very large (or very small) can form chains Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 mitosporic fungi often responsable of: biodeterioration biotechnology (enzymes, hormones, etc production) food industry (beer, wine, cheese, …) pharmaceutical (allergen extract., antibiotic prod., …) mycoses mycotoxins allergies infections (A. fumigatus) sick building syndrom often associated with moisture and molds plant pathogens Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 Fungal allergens not enough data and research one problem: lack of standardized extracts well known: Cladosporium (in particular children) Alternaria (and other cross-reactive) Stachybotris (indoor, big story in the USA) occupational allergies in the mushroom industry (eg Pleurotus) Michel Thibaudon – March 2018

Michel Thibaudon – March 2018

How to collect fungal spores Material and method 2 How to collect fungal spores Material and method Michel Thibaudon – March 2018

Today, we will discuss two very important topics in aerobiology: viable spores sampling, problem of the substrate (growing medium) … indoor and outdoor aerobiology Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 Outdoor fungal spores: Hirst method - Continuous detection Michel Thibaudon – March 2018

vocal recognition system 1 1 Outdoor fungal spores: Hirst method - Continuous detection Dividing the band into daily sections 1 slide by day Counting with a vocal recognition system Collection of the drum band spores count Michel Thibaudon – March 2018

1 Cyclonic method Coriolis Specific or non-inert particles optical microscopy Coriolis Coloration / filtration Easy and rapid method Air Liquid Particles 300 l/min 3 m3 in 10 min Liquid sample for rapid analysis PCR Method for specific identification Chemscan Transfert of biological particles from the air to a collect LIQUID medium BROADBAND LAL Endotoxins Mycotoxins Method with high sensibility Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 Impaction on culture medium Culture Culture Culture Be careful to the quality of culture medium Michel Thibaudon – March 2018

3 Filtration method Incubation Transfer gelatin membrane on petri dish Air filtration on dense gelatin membrane Incubation Be careful to the quality of culture medium Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 4 Continious impaction : pollens and molds trap : LANZONI VPPS 2000 Impaction on culture medium : MAS 100 Filtration on gelatin membrane : AIRScan Cyclonic sampler : Coriolis®µ Equipment used for this study: Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 4 Analysis methods used: Culture medium : Trypto casein soy, Sabouraud MEA Incubation temperatures between 20°C and 25°C. Identifications : Macroscopic Microscopic by transfer on transparents supports, coloration and placed on glass slides. Numeration realised when it is possible (invasions limiting quality counts in some samples) Michel Thibaudon – March 2018

4 Analysis methods used: Detection method by optical microscopy (for liquid samples from cyclonic sampler and gelatin membranes membranes gélatines whole or fractionned) : Analysis by optical microscopy, magnification x400 4 Analysis methods used: Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 4 Spore Identification Important Characteristics Color Size Shape Septation Attachments Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 4 Study period: Sampling from 2010 to 2012 in very different environments (during 3 years): Indoor air Industries facilitise Offices Hotel House Laboratories Clean room Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 3 Results Michel Thibaudon – March 2018

3 Results Moulds Hirst trap MICROSCOPIC OBSERVATION Impaction / filtration on dense gelatin membrane Culture on : Cyclonic trap/ filtration on dense gelatin membrane MICROSCOPIC OBSERVATION TSA Sabouraud MEA Absidia + ++ +++ Acremonium Alternaria Ascospores Aspergillacea Aspergillus Penicillium Aureobasidium Basidiospores Botrytis Cercospora Chaetomium Cladosporium Didymella Entomophthora Epicoccum Erysiphe Fusarium Fusicladium Helicomyces Helminthosporium Mucorales Myxomycetes Easy detection Possibility of detection No possibility of detection Michel Thibaudon – March 2018 +++ ++ +

3 4 Results Moulds Hirst trap MICROSCOPIC OBSERVATION Impaction / filtration on dense gelatin membrane Culture on : Cyclonic trap/ filtration on dense gelatin membrane MICROSCOPIC OBSERVATION TSA Sabouraud MEA Peronospora +++ Pithomyces + ++ Pleospora Polythrincium Sporidesmium Sporobolomyces Stemphylium Stachybotrys Taeniolella Tilletiopsis Torula Trichoderma Trichothecium Uredospores Ustilago Easy detection Possibility of detection No possibility of detection Michel Thibaudon – March 2018 +++ ++ +

3 4 Results Air contamination Culture Optical microscopy detection Spores small quantity ++ +++ Detection + Counting Identification Spores high quantity Détection ++ (invasion problem) Mycelium small quantity Mycelium high quantity Easy detection Possibility of detection No possibility of detection Michel Thibaudon – March 2018 +++ ++ +

Michel Thibaudon – March 2018 3 4 Results Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 3 4 Results Moulds Infectious impacts Allergic impacts Alveolit Toxic impacts Absidia corymbifera YES Acremonium NO DATA Alternaria alternata Aspergillus flavus Aspergillus fumigatus Aspergillus glaucus Aspergillus niger Aspergillus versicolor Aureobasidium Chaetomium Cladosporium sphaerospermum Epicoccum Fusarium Mucorales Penicillium sp. Stachybotrys chartarum Trichoderma Trichothecium Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 3 4 Results Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 3 4 Results Outdoor fungal spores 2017 Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 3 4 Results Outdoor fungal spores 2017 Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 3 4 Results Outdoor fungal spores 2017 Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 3 4 Results Michel Thibaudon – March 2018

Example of recommendation 4 Example of recommendation Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 3 4 Results We must destroy the wall Pollens Moisissures Bactéries Virus Air intérieur Extérieur Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 4 4 Seasonality and risk of contamination Implentation of Action plan Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 5 Conclusion Michel Thibaudon – March 2018

Michel Thibaudon – March 2018 All methods of sampling and analysis based on detection thanks to culture of molds viable and culturable, leave aside a considerable number of non- culturable fungi. It is important to associate classic analysis culture and methods of analysis by direct detection (optical microscopy) and qualitative methods (PCR…) The future development of standards, including the ISO 14698, must consider these issues on the mastery of fungal contamination in cleanrooms Michel Thibaudon – March 2018

Thanks for your attention Happy end Thanks for your attention Michel Thibaudon – March 2018