The Complexity of Damp Indoor Spaces Jack D. Thrasher, Ph.D. Sandra Crawley, M.ED., LADC Michelle DeFazio, BSAT – NTEF #1

Introduction Water Intrusion – fungi, bacteria and protozoa Multiple Biocontaminants Additive and Synergistic Effects Increased Health Risks #2

Additive and Synergistic Effects It is apparent that additive and synergistic effects have been demonstrated in vitro and in vivo. 1.Vomitoxin and LPS increase apoptosis in lymphoid organs and proinflammatory cytokines in murine models 2. Roridin A and LPS – potentiates neurotoxicity in nasal airways of mice 3.Aflatoxin B1 and LPS – synergistic toxicity to liver#3

References for Introduction and Public Health Anderson et al (1997), Appl Environ Microbiol, 63: Peltola et al (2001a,b) Appl Environ Microbiol 67: ; Hirvonen et al (2005) Indoor Air 25(suppl9): Rintala et al (2001) Lett Appl Microbiol 23: Fisk et al (2007) Indoor Air 17: Mudarri et al (2007: Indoor Air 17: Genuis SJ (2007) Eur J Intern Med 28: Bernstein, et al J Allergy Clin Immunol 91:585:91.#4

References for Introduction Cont. LPS AND MYCOTOXIN SYNERGISM Islam Z et al (2002) Toxicol Appl Pharmacol 280: Islam Z et al (2007) Toxicol Sci 98: Islam Z, Pestka JJ (2006) Toxicol Appl Pharmacol 26: Luyendyk JP et al (2003, 2003). Toxicol Sci 68:220-5; 72: Baron CC, et al (2001) Hepatology 33:44-73 #5 Toxicol Sci 98:

Water Intrusion Water Activity (Aw) – Water Content The ratio of vapor pressure exerted by water in the material to the vapor pressure of pure water at the same Temp. and Pressure #6

INDOOR BIOCONTAMINANTS Indicator molds Bacteria- Gram (+) and (-) Particulates: Large & Small (<2 micron) Mycotoxins Exotoxins #7 MVOCs Enzymes (Allergens) Hemolysin & Siderophores Galactomannans 1-3 beta-Glucans Endotoxins

Molds in Relation to Water Activity This table summarizes the key water activity (aw) and molds that grow most readily as primary, secondary and tertiary colonizers (adapted from Nielsen, 2003; Gorny, 2004). Primary colonizers (aw <0.85): Alternaria citri, Aspergillus (Eurotium) amstelodomi, Aspergillus candid\us, glaucus, niger, penicilloides, repens, restrictus, versicolor; Paedilomyces varlotti, Penicillium aurantiogriseum, brevicompactum, chrysogenum, commune, expansum, griseoflavum; Wallemia sebi Secondary colonizers (aw = 85-90): Cladosporium cladosporoides, herbarum, sphaerosperumum; Mucor circinelloides; Rhizopus oryzae. Tertiary colonizers (aw = >90): Alternaria alternata; Aspergillus fumigatus; Epicoccum species; Exophiala species; Fusarium moniforme; Mucor plumbeus; Phoma herbarum; Phiaosphora species; Trichoderma species; Stachybotrys chartarum; Ulocladium consortiale; Rhodotorula species, Sporobolomyces species; and Actinomyces (Actinobacteria). #8

Indicator Molds Aspergillus fumigatus, versicolor, flavus, niger, sydowii, amstelodomi Penicillium brevicompactum, chrysogenum, chrysogenum Trichoderma sp. Fusarium moniforme. Epiccocum sp. Stachybotrys chartarum (two chemotypes) #9

Identification of Molds PCR DNA to determine species Viable air samples Bulk samples Wipe Samples Wall Cavity Samples Compare species indoors vs outdoors. If Aspergillus, Penicillium. Stachybotrys & Actinomycete species are above outdoors - Contamination Exists #10

Particulates – Indoor Air Two Groups of Particulates 1.Spores and Hyphae at 2 microns and up. 2.Fragments <2 microns. #11

Fine Particulates 1.Gorny (2004) Filamentous microorganisms and their fragments in the Indoor Air: A review. Ann Agric Environ Med 11: Gorny et al (2002) Fungal Fragments as Indoor Air Biocontaminants. Appl Environ Microbiol Gorny et al (2003) Release of Streptomyces albus propagules from contaminated surfaces. Environ Res 91: #12

Summary of Gorny’s Work Particulates < 2 microns are aerosolized as fragments from mycelia and spores. 320 times more concentrated than spores. Low Air velocity and vibration cause the release of these fragments. The vibrations of 1-20 hertz with 1 hertz most effective – normal human activity. Fragments contain allergens, enzymes, glucans, galactomannans, mycotoxins, etc. #13

Fine Particulates and Trichothecenes Brasel, et al (2005) Detection of airborne Stachybotrys chartarum macrocyclic trichothecene mycotoxins in the indoor environments. Appl Environ Microbiol 71: Brasel, et al (2005) Detection of airborne Stachybotrys chartarum macrocyclic trichothecene mycotoxins on particulates smaller than conidia. Appl Environ Microbiol 71: Brasel, et al (2004) Detection of trichothecene mycotoxins in sera from individuals in Stachybotrys chartarum indoor environments. Arch Environ Health 59: Gottschalk C, et al (2008) Detection of Satratoxin G and H in indoor air from a water-damaged building. Mycopathologia 166: Van Emon, et al (200) ELISA measurement of stachylysin in serum to quantify human exposure to the indoor mold Stachybotrys chartarum. J Occup Environ Med 45: #14

Summary of Brasel and Van Emon Papers Homes contaminated with Stachybotrys and multiple health complaints. Tested for Trichothecenes in contaminated vs non- contaminated buildings and outdoors. Multistage filtration: 5.0, 1.2 & O.4 microns, SEM, HPLC, ELISA. Buildings contained from 1,300 ug/m 3. Difference between experimental and control building, p <0.001 Sera testes: n = 44 (contaminated; n = 26 (uncontaminated). GC/MS. The exposed had significantly higher concentrations in sera vs controls (p 0.001) Stachylysin: sera of rat pup with nasal installation and 5 adult males in a contaminated environment. Stachylysin averaged 371 mg/ml of serum. #15

Indoor Mycotoxins Cont. Smoragiewicz, et al (1993) Trichothecene mycotoxins in the dust of ventilation systems in office buildings. Int Arch Occup Environ Health 65: Engelhart, et al (2002) Occurrence of toxigenic Aspergillus versicolor isolates and sterigmatocystin in carpet dust from a damp building. Appl Environ Microbiol 68: Chapin-Kadouch et al (2006)Mycotoins identification in moldy dwellings. J Appl Toxicol 26:475-9 Bloom et al (2007) Mass spectrometry-based strategy for direct detection and quantification of some mycotoxins produced by Stachybotrys chartarum and Aspergillus spp in indoor environments. Appl Environ Microbiol 73: #16

Ergosterol and Respiratory Illness Park et al (2008) Hydrophilic fungi and ergosterol associated with respiratory Illness. Environ Health Perspec 116: n = 200 with 123 respiratory disease = asthma Asthma was significantly associated with presence of hydrophilic and Ergosterol. Ergosterol is a sterol found in fungal walls. #17

Aspergillosis and Gliotoxin Lewis et al (2005) Frequency and species distribution of gliotoxin producing Aspergillus isolates recovered from patients at a tertiary-care cancer center. J Clin Microbiol 43: Lewis et al (2005) Detection of gliotoxin in experimental and human aspergillosis. Infect Immunity 73: Gliotoxin was produced by isolates: fumigatus (93%); niger (75%), terreus (25%) flavus (4%). 2. Mice: Lungs (m = 3,975 ng/g; Sera (M = 36.9 ng/ml) Human Sera: 2 of 11 patients without confirmed I.A. In Confirmed I.A. patients 4 of 5 (range 166 to 785 ng/ml). #18

Bacteria Gram Negative and Positive – Examples – Gorny & Dukiewicz, 2002; Bouillard et al, Gram Negative Enterobacteriaceae Pseudomonas sp. Proteus sp. Actinobacter sp. Alcaligenes faecalis (aerobes and anaerobes) #19 Gram Positive Micrococcus sp. Bacillus sp Streptococcus sp Enterococcus sp. Mycobacteria – Streptomyces sp, Nocardia sp., Mycobacterium sp.

Endotoxins - Indoor Chew et al (2006) Mold and endotoxin levels in the aftermath of hurricane Katrina: A pilot project of homes in New Orleans. Environ Health Perspec 114: Rao et al (2007) Characterization of airborne molds, endotoxins, and glucans in the homes of New Orleans after hurricanes Katrina and Rita. Appl Environ Microbiol 73: Gorny, Dutkiewcz (2002) Bacterial and fungal aerosols in indoor environment in Central and Easter European countries. Ann Agric environ Med. 9:17-23 Gorny (2004) Filamentous microorganisms and their fragments in the Indoor Air: A review. Ann Agric Environ Med 11: #20

Endotoxins – Indoor Air 1. Inhaled LPS are associated with inflammation of the lungs involving TNF-alpha, and activation of C3. 2. There is complicated interaction between TLR4 receptors and CD14 polymorphism – CD14-CC and CD14-TT including carrier proteins M88 and TRAM 3. IgE interactions are not the norm. IL4, IL5, eosinophils and neutrophils are involved and difficult to control. 4.CC phenotypes react to low concentrations, while TT carriers react to higher concentrations of endotoxins – Wheeze not Asthma #21

CD14 and TLR4 - Endotoxins 1.Martinez (2007) CD14, endotoxin and asthma. Proc Amer Thoracic Soc. 4a; Martinez (2007) Gene-environment interactions in asthma. Proc Amer Thoracic Soc 4: Simpson et al (2006) Endotoxin exposure, CD14, and allergic diseases. Am J Respir Crit Care Med 174: Sackesen et al (2005) The effect of polymorphism at the CD14 promoter and the TLR4 gene on asthma phenotypes in Turkish children with asthma. Allergy 60: #22

Actinomycetes Streptomyces – Up to 13 different species Nocardia, at least two species Mycobacterium – Several different species. #23

Streptomyces and Toxicity Three Key Papers Huttunen et al (2004) Synergistic interaction in simultaneous exposure to Streptomyces californicus and Stachybotrys chartarum. Environ Health Perspec 112: Andersson et al (1998) Mitochondrial poison by Streptomyces griseus strains isolated from an indoor environment is Valinomycin. Appl Environ Microbiol 12: Jussila, et al (2003) Systemic immune responses in mice after repeated exposure of lungs to spores of Streptomyces californicus. Clin Diag Lab Immunol 10:30-7 #24

Summary of Streptomyces Papers Synergistic action occurs with trichothecenes and S. californicus with respect to IL-4, TNF-alpha in vitro. Vanlinomycin – caused mitochondrial swelling, inhibiting oxidative phosphorylation and motility – boar sperm cells. Spores of S. californicus cause inflammation in the lungs and systemic immunotoxicity in the spleen and other lymphoid organs. #25

Toxic Nocardia Peltola et al (2004) Isolation of toxigenic Nocardia strains from indoor environments and description of two new Nocardiopsis species, N. exhalans sp. nov. and N. umisdischolae sp. nov. Appl Environ Microbiol 67: Methanol soluble toxin that depleted ATP sperm cells. 2. Destruction of Cell membrane of sperm cells. 3. The toxic properties suggests good survival in the indoor environment. #26

Mycobacterium – Non-Tuberculin Torvinen et al (2000) Mycobacteria and fungi in moisture- damaged building materials. Appl Environ Microbiol 72: Jussila et al (2002) Mycobacterium terrae isolated form indoor air of a moisture-damaged building induces biphasic inflammatory response in mouse lungs. Environ Health Perspec 110: Falkinham (2003) Mycobacterial aerosols and respiratory disease. CDC. Emerging Diseases. 9(No. 7) July American Thoracic Society. (1997) Diagnosis and treatment causes by nontuberculous mycobacteria. Respir Crit Care Med 256:S1-S55.#27

Concerns of Nontuberculin Mycobacteria Torvinen – Nontuberculin mycobacteria are present in water-damaged building materials. American Thoracic Society – Nontuberculin Mycobacterium infections are on the increase worldwide. The cause is unknown. Falkinham – Mycobacterium terrae, immunogenum and avium have been implicated in outbreaks of Hypersensitivity pneumonitis. Jussila et al – Two phase inflammatory response to M. terrae. –A. Initial response lasts 6 hours to 3 days- TNF alpha & IL-6 and neutrophils and increased LDH. –B, Second Phase last up 7-28 days – increase production of TNF- alpha and Nitric Oxide synthase, mononuclear cells infiltration and increased LDH. #28

Hemolysins In Cleveland, Ohio, pulmonary hemosiderosis was initially blamed on S. chartarum, Stachylysin and a siderophore produced by S. chartarum. Vesper and Vesper recently published data on hemolysins produced by Aspergillus, Penicillium and other molds isolated from the Cleveland Homes. Advances Appl. Microbiol, V 55. A. 12 species of Aspergillus B. 11 species of Penicillium. C. Others – Emericella, Memnoniella, Trichoderma species #29

Other Irritants and Toxins 1,3-Beta glucans Galactomannans MVOCs Time does not allow us to go into full details of these contaminants. #30

Olfactory Tract and Mycotoxins Larrson, Tjaive (2000) Intranasal instillation of Aflatoxin B1 in rats: Bioactivation in the nasal mucosa and neuronal transport to the olfactory bulb. Toxicol Sci 55: Islam et al (2006) Satratoxin G from the black mold Stachybotrys chartarum evokes olfactory sensory neuron loss and inflammation in the murine nose and brain. Environ Health Perspec 114: Islam et al (2007) Neurotoxicity and inflammation in the nasal airways of mice exposed to the macrocyclic trichothecene mycotoxin roridin a: kinetics and potentiation by bacterial polysaccharide coexposure. Toxicol Sci 98: Shwe TTT et al (2008) Changes in neurotransmitter levels and proinflammatory cytokine mRNA expressions in the mice olfactory bulb following nanoparticle exposure. Toxicol Appl Pharm 226: #31

Air Pollution and Olfactory Tract- Humans Calderon-Garciduenas (2004) Brain inflammation and Alzheimer’s-like pathology in individuals exposed to severe air pollution. Toxicol Pathol 42: Calderon-Garciduenas (2008) Long-term air pollution exposure is associated with neuroinflammation, innate immune response, disruption of the blood-brain barrier, ultrafine particulate deposition and accumulation of amyloid beta-42 and alpha-synuclein in children and young adults. Toxicol Pathol 36: Calderon-Garciduenas et al (In press) Air pollution: cognitive deficits and brain abnormalities: a pilot study with children and dogs. Brain and Cognition #32

Conclusions 1.As we have seen here today, the indoor environment resulting from microbial growth in response to water intrusion is very complex involving molds, bacteria and their by-products. 2.The interactions of the by-products are complex and probably include synergism 3.The biocontaminants of the indoor environment should be systematically tested for mold species, bacterial species as well as mycotoxins. 4.Air, bulk, wipe and wall cavity samples with PCR-DNA analysis of mold and bacterial species #33