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Indoor Molds and Mycotoxins

Indoor Molds and Mycotoxins. Estelle Levetin, PhD Faculty of Biological Science University of Tulsa. Fungi. Abundant in the natural environment and able to grow on many environmental and synthetic substrates Capable of producing secondary metabolites, mycotoxins and VOCs

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Indoor Molds and Mycotoxins

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  1. Indoor Molds and Mycotoxins Estelle Levetin, PhD Faculty of Biological Science University of Tulsa

  2. Fungi • Abundant in the natural environment and able to grow on many environmental and synthetic substrates • Capable of producing secondary metabolites, mycotoxins and VOCs • Small percent are plant or animal pathogens • In terms of human exposure, fungi can be responsible for allergic, infectious, or toxic diseases

  3. Major Groups of Fungi • Myxomycetes - slime molds • Oomycetes - water molds • Zygomycetes - bread molds, sugar fungi • Ascomycetes - sac fungi, morels, yeast • Basidiomycetes - mushrooms, puffballs • Asexual fungi (Deuteromycetes)- molds, microfungi, anamorphic fungi, mitosporic fungi – asexual stages of ascomycetes

  4. Fungal Spores • Fungi reproduce by spores • Majority of spore types adapted for airborne dispersal • Spores unicellular to multicellular from 1 to 100 m – always microscopic

  5. Outdoor Fungal Spores • Amazing diversity of spores in atmosphere • Concentrations up to 200,000 spores/m3 • Daytime dominated by “Dry Air Spora” Cladosporium, Alternaria, Drechslera, Curvularia, Pithomyces, andsmut spores • Late night and early morning dominated by basidiospores and ascospores • Rainy periods dominated by ascospores

  6. Fungal Spores in Outdoor Air

  7. Mold Spores in the Indoor Environment • Spores occur in all indoor environments • Considered contaminants indoors • Fungi can colonize countless substrates indoors • HVAC system can also become contaminated and even help disperse spores

  8. Environmental factors that influence indoor fungal contamination • Outdoor concentration and type • Type and rate of ventilation • Activity levels • Modern building materials • Indoor moisture levels

  9. Typical Yearly Spore Levels

  10. Environmental factors that influence indoor fungal contamination • Outdoor concentration and type • Type and rate of ventilation • Activity levels • Indoor moisture levels • Modern building materials

  11. Environmental factors that influence indoor fungal contamination • Outdoor concentration and type • Type and rate of ventilation • Activity levels • Indoor moisture levels • Modern building materials

  12. Environmental factors that influence indoor fungal contamination • Outdoor concentration and type • Type and rate of ventilation • Activity levels • Indoor moisture levels • Modern building materials

  13. Indoor Moisture Levels • Availability of moisture most critical factor in determining if fungi will grow • Leaks and moisture seeping through walls, ceilings, basements obvious source of problems

  14. Courtesy of Terry Brennan

  15. Moisture Problems • Become worse in past 20 - 30 yrs • increased use of washing machines, dishwashers • vaporizers and humidifiers actively spray droplets into the air (often contaminated) • tighter buildings for energy conservation trap moisture • Anytime moisture available fungi will grow

  16. Humidity • Indoor relative humidity • Below 30% R.H. no mold growth and • Above 70% optimal for mold • Usually above 50% mold growth can occur • Humid air condenses on cool surfaces • Cold windows in winter - molding and sills become wet and suitable for fungal growth • Cold floors in winter • Cooling coils in AC units in summer • Humid air allows hygroscopic materials to absorb water

  17. High humidity in home where subslab ducts failed

  18. Environmental factors that influence indoor fungal contamination • Outdoor concentration and type • Type and rate of ventilation • Activity levels • Indoor moisture levels • Modern building materials

  19. Modern Building Materials • Ceiling tiles, sheetrock, and other building materials seem to be especially prone to fungal contamination • High cellulose content • Many fungi are cellulose degraders in the natural environment - perfect substrate to culturing fungi

  20. Objective of Study • What happens when new ceiling tiles are soaked with water? • How long does it take before contamination is visible? • What fungi are present?

  21. Methods • New ceiling tiles aseptically cut into 6 x 6 cm squares and placed in sterile petri dishes • Ceiling Tile Squares (CTS) saturated with 33 ml water: • sterile distilled water • tap water • rain water • CTS in triplicate, experiment repeated, and extra sterile distilled water saturated CTS

  22. Methods • Dishes sealed with parafilm and allowed to incubate at room temperature for a minimum of 10 weeks • CTS were regularly evaluated by direct microscopy of surface growth • At the end of the experiment randomly selected CTS were ground up and cultured

  23. Direct Microscopy • CTS within sealed petri dishes were evaluated with a dissecting microscope • Fungi were identified • After 10 weeks estimates made of percent surface area covered by fungal growth

  24. Results of Direct Microscopy • All CTS showed fungal contamination • CTS saturated with tap-water had the greatest contamination had a mean surface coverage 65% (range 50-90%) • CTS saturated with sterile distilled water had a mean coverage of 60%(range 5-100%) • CTS saturated with rainwater showed the lowest contamination with a mean coverage of 10% (range 1-25%). • 10 genera of fungi identified plus yeast

  25. Fungal Growth on CTS after 10 Weeks Incubation Rainwater Sterile Distilled Water Tap Water

  26. Alternaria Aspergillus Chaetomium Cladosporium Drechslera Epicoccum Mucor Penicillium Stachybotrys Ulocladium Yeast Fungi Identified by Microscopy

  27. Additional Observations • Visible colonies of Alternaria and Epicoccum appeared within 4 days after water was added • Other fungi appeared much later • After 10 weeks incubation, dominant fungal types of CTS varied greatly • Alternaria dominant on some • Stachybotrys dominant on some • Chaetomium dominant on some

  28. Culture Analysis • One CTS was randomly selected from each “water” type from each experiment • 2 sterile distilled water saturated CTS • 2 tap saturated CTS • 2 rainwater saturated CTS • Three additional CTS from sterile distilled water group also selected at random

  29. Culture Analysis Methods • Each CTS was placed in a sterile blender cup with 50 ml of sterile distilled water • CTS blended on high for two 15 sec intervals • Resulting slurry was filter through 4 layers of sterile cheese cloth • Resulting suspension was dilution plated on MEA + strep and Cellulose Agar • Incubated at room temp for 7 to 10 days

  30. Results of Culture Analysis • Overall Penicillium most abundant genus on culture plates - found on all “water” types • Several fungi that were dominant on tile surfaces were low or absent in culture • Quantification difficult when plates overgrown with Penicillium • Results of all media and all dilutions combined

  31. Summary of Fungi Identified on CTS

  32. Indoor Fungi • Indoor spores generally reflects outdoors unless there is a source of contamination • Many different types of fungi occur - 160 spp • Most common genus is Cladosporium - just like it is outdoors – some species difference indoors • BUTPenicillium and Aspergillus often exist at higher concentrations indoors • Stachybotrys has received most media attention over the past 4 years

  33. Sampling Andersen (N-6) sampler Various portable spore trap impactors

  34. Cladosporium • Common fungal genus occurring both indoors and outdoors • Most abundant outdoor spore type with a worldwide distribution • Normally exists as a saprobe or weak plant pathogen • Spores are known to be allergenic

  35. Cladosporium spp.

  36. Cladosporium on diffuser

  37. Penicillium • One of the most common soil fungi in natural environment • Over 250 species • Well known allergen • Some species produce mycotoxins • Some species produce antibiotics • Produce VOCs

  38. Aspergillus • Also common soil fungi • Cause rot of stored grain • Over 150 species • Well known allergens • Several species form mycotoxins • Some species can grow at high temperatures • Several species cause infections in lung, sinuses, and hypersensitivity pneumonitis

  39. Penicillium and Aspergillus • Small spores passively aerosolized when spore clusters disturbed • Spores extremely buoyant, remain airborne for extended time • Penicillium and Aspergillus spores look alike – distinguished in culture

  40. Pen/Asp Concentrations • Previous and on-going studies in my lab have focused on trying to determine base-line levels of Penicillium/Aspergillus levels • Collected multiple air samples from 12 indoor locations during Sept, Nov, Feb along with outdoor controls • Andersen samples for culturable fungi • Spore trap samples for total spores

  41. Pen/Asp Concentrations • Penicillium and Aspergillus identified in all locations • Culture analysis identified • 23 species of Penicillium (mean 39.1 CFU/m3) • 12 species of Aspergillus (mean 14.1 CFU/m3) • Spore trap samples found • 332 spores/m3 as the mean level of Penicillium/Aspergillus type spores indoors

  42. Stachybotrys chartarum • Soil fungus in nature • Commonly found indoors on wet materials containing cellulose, such as wallboard, jute, wicker, straw baskets, and paper materials • Spores in slimy mass • Thought to be allergenic although little is known • May produce potent mycotoxins

  43. Stachybotrys

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