Biomarkers of Exposure to Microbial Contaminants Papers (251-257)

251. The Use of Ergosterol to Measure Exposure to Fungal Propagules in Indoor Air.

J. Miller, J. Young, Plant Research Centre, Ottawa, Ontario, Canada

The growth of fungi in buildings leads to health and comfort complaints depending on the fungi involved, duration of exposure and individual sensitivity. Fungal exposures in indoor air are usually estimated by the collection of air samples by impaction on agar surfaces. Such samples provide information on "culturable" propagules but greatly underestimate the total spore numbers present. there are several reasons for this including decline in spore viability, choice of agar medium, and damage to the spores during sampling. Several comparative studies have reported positive correlations between viable and total fungal spores estimated by direct microscopy of microporous filter samples, but this varies according to the spore concentrations, which are subject to extreme temporal variation. Although reliable qualitative information is obtained, no useful quantitative information is acquired from such measurements. The alternative approach is to measure an indicator biochemical present in the fungal propagules. One such chemical is -1,3-glucan, however, no studies have been reported on the variability of the concentration of this chemical in fungal spores. Another possibility is the fungal sterol ergosterol, which has been widely used in fungal ecology studies to estimate fungal biomass.

Ergosterol was determined in spores of 11 species of Aspergillus, Penicillium and Cladosporium selected from the most common molds in 400 homes in Ontario. The ergosterol content of spores was highly correlated to their volume (r=0.785, p<0.007). Spore ergosterol content was ca. 1 g/mg, which is the range reported for mycelia and varied by ca. 25% for the species tested. Ergosterol was determined in bedroom air samples taken during the winter in homes in southern Ontario. The median ergosterol value corresponded to a total concentration of fungal spores of ca. 100 per m, in the range for other studies where total and viable propagules were determined by other methods. The sampling of air for ergosterol is a robust method for assessing fungal biomass in air.

This method has been extended to settled dust which contributes to the fungal burden of air.

252. Ergosterol and Its Decomposition Products in Animal Feed Aerosols.

M. Strength, AT&T, Atlanta, GA; P. Gao, H. Dillon, University of Alabama at Birmingham, Birmingham, AL

Ergosterol has been proposed as an index of fungal biomass in a variety of bulk biogenic materials and in house dust. The purpose of this study was to examine the relationship between fungal propagule count and ergosterol and photolytic decomposition products of ergosterol in dust suspended during the preparation of animal feed. Relatively large volumes of air (5-20 m) were sampled through Teflon-coated glass fiber filters. The collected aerosols were analyzed by C18 reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection at a wavelength of 282 nm, by C8 reversed phase HPLC with tandem mass spectrometric detection (MS/MS) with an electroscopy interface, and by microscopy using bright field illumination with computer-assisted image analysis to determine a count of fungal propagules.

A regression of the mass of ergosterol found by HPLC/UV against fungal spore count yielded as statistically significant (p<0.025) correlation coefficient of 0.92 (n=5). Analysis by HPLC/MS/MS indicated the presence of not only ergosterol but two photolytic degradation products, Vitamin D2 and either tachysterol or lumisterol. The total amounts of these compounds and ergosterol correlated with the amounts of ergosterol observed by HPLC/UV (r= 0.84, p<0.05, n=5) but were estimated to be an order of magnitude higher.

It was concluded that either ergosterol alone or the sum of ergosterol and accompanying decomposition products provided an index of fungal propagule count in a specific aerosol generated by the mixing of animal feed. The co-occurrence of these degradation products with ergosterol may provide additional useful information about the "aging" of fungal contaminants in biogenic dusts.

253. Toxins Produced by Fungi Found in Water-Damaged Buildings.

W. Sorenson, NIOSH, Morgantown, WV; B. Jarvis, J. Jiang, Y. Zhou, University of Maryland, College Park, MD

Mycotoxins are small molecule secondary metabolites produced by a wide range of fungi which pose a risk to the health of humans and animals. Numerous toxigenic fungi are well known to contaminate food and feed and thus pose a hazard through ingestion of farm produce. What is less well appreciated is that water-damaged buildings can also harbor toxigenic fungi which threaten the health of the building occupants. Since the vast majority of mycotoxins are not volatile, the principal source of exposure in these buildings is due to inhalation of aerosolized spores and fungal-contaminated building materials. Our present state of knowledge suggests that only a relatively small number of fungal species (e.g., various Penicillium, Aspergillus, Fusarium, and especially Stachybotrys atra) pose a general health hazard. Most reported cases deal with work-related disease, e.g., increased risk of liver cancer in granary workers exposed to aflatoxin-contaminated dust, although there is increasing concern with fungi in damp homes.

In the winter of 1994, a cluster of 14 cases of pulmonary hemosiderosis was reported in the eastern part of Cleveland, Ohio. These unusual cases appeared only in infants ranging in age between one and eight months and were characterized by massive bleeding in the lungs. No infective agents or environmental toxins were shown to be associated with this syndrome. However, all the target homes had continuing water problems which were most severe in the preceding summer when Cleveland experienced serious flooding in the area. The toxigenic fungus, Stachybotrys atra was found in all but one of the homes, and the levels of fungal activity were considerably higher than those found in control homes. S. atra isolates collected from these homes have been shown to produce a number of highly toxic compounds.

254. Monitoring of Bacterial Load Using Gas Chromatography-Tandem Mass Spectrometry for Muramic Acid Analysis and Culture Techniques.

A. Fox, K. Fox, M. Krahmer, University of South Carolina, Columbia, SC

Plating techniques are traditionally used for assessing viable bacteria numbers in air. Alternatively, muramic acid, as a marker for peptidoglycan (PG) allows assessment of total bacterial load (including both culturable, non-culturable, dead and degraded cells). PG is present in both gram positive and gram negative bacterial PG and mimics the biological activity of endotoxin in many systems. Samples for plating were collected in a barn using a single stage Andersen sampler. Colonies were characterized morphologically and by gram stain. The mean bacterial load was 2.4 x 10 colony forming units/m of air. The majority of colonies were gram-positive bacilli and filamentous rods. Airborne dust was also collected on Teflon filters for MA analysis. The entire filter and associated dust was hydrolyzed to release MA which was converted to alditol acetate derivative and analyzed by gas chromatography-tandem mass spectrometry (GC-MS-MS). Most of our experience has been with triple quadropole instrumentation. Although, encouraging results have been obtained with a more modestly priced ion trap MS-MS instrument. The concentration of MA in air was 1.6 0.2 ng/m. This value translates to a total bacteria load of 6.5 x 10 (if the non-culturable population was indeed primarily gram-positive) or 1.7 x 105 bacteria/m (if the non-culturable population was primarily gram-negative). Emission of muramic acid values (as a measure of bacterial peptidoglycan), using tandem mass spectrometry, adds to the limited group of indirect tests available for assessing airborne biocontamination.

255. Quantification of Lipopolysaccharide and Fungal Biomass in Organic Dust by Gas Chromatography-Mass Spectrometry.

L. Larsson, University of Lund, Lund, Sweden; H. Burge, D. Milton, Harvard School of Public Health, Boston, MA

Lipopolysaccharide (LPS) from gram-negative bacteria and certain fungi exhibit toxic effects upon inhalation. Determination by gas chromatography-mass spectrometry (GC-MS) of 3-hydroxy fatty acids (3-OH FAs), which are LPS-specific constituents, and ergosterol, which is a fungus-specific membrane lipid, have been suggested as alternatives to the use of Limulus-based bioassay methods for measuring potency of endotoxin (the toxic component of which is LPS) and beta 1,3 glucan (present in many fungi). A GC-MS method for determining both 3-OH FAs and ergosterol from the same dust is described in the following.

Samples of organic dust are heated in methanolic KOH and extracted with hexan:water. The organic phase is evaporated, purified, and subjected to trimethylsilyl (TMS) derivization of ergosterol. The aqueous phase is acidified, evaporated, heated in methanolic HCl, extracted, purified, and subjected to TMS derivization of the 3-OH FAs. GC-MS analysis has revealed that >95% of each of the 3-OH FAs present in the dust samples is found in the aqueous layer. The relative amounts of individual 3-OH FAs in the samples provides information about the gram-negative bacteria present. By using tandem MS (GC-MSMS), a method that has recently become practical due to developments in instrumentation, an even higher detection specificity is achieved than with conventional GC-MS.

The amounts of 3-OHFAs in dust samples can be correlated with endotoxin activity and the amounts of ergosterol correlated with culturable fungi. The described method may be used for correlating inhalable LPS and fungal biomass exposure with respiratory symptoms and pulmonary function changes.

256. Detecting Non-Culturable Air Biocontamination: Direct Quantitative Sampling of Indoor Air Biomass by Lipid Biomarker Analysis.

S. MacNaughton, Microbial Insights, Inc., Knoxville, TN

To a large extent, standard methods for characterizing the microbial community of indoor air rely on culture based techniques such as Andersen, impactor, or impinger sampling. The success of these classical techniques depend on the ability of an organism to grow on "non-selective" media. However, as has been repeatedly documented, such viable counts consistently underestimate bacterial numbers from environmental samples by between 90-99.9%.

One approach to ameliorating the problems associated with culturing techniques is to determine biomass via the analysis of specific biochemical cell components. Lipids are major components of all cell membranes, are labile and have profiles that vary considerably dependant on microorganism species. To test the hypothesis that lipid analysis will enable quantification of bioaerosols, it was first necessary to compare lipid analysis of known volumes of culturable test organisms with conventional sampling method. Replicate samples of Escherichia coli, Bacillus subtilis and Mycobacterium fortuitum and two mixtures of these organisms were deposited onto filtration media using a test stand constructed as a modification of the ASTM 1215 standard. Filter deposited bacterial numbers were determined by two methods: 1) by viable counts on media following collection in an all glass liquid impinger; and 2) by phospholipid analysis. Lipids were extracted from organisms on the filtration media in chloroform: methanol: buffer solvent from which the lipids were recovered in the organic phase. Phospholipids were separated out from the extracts by fractionation on silicic acid columns, methylated, profiles analyzed and specific peaks identified by gas-chromatography/mass-spectrometry (GC-MS). Lipid biomarker analysis of the bacterial phospholipids showed that numbers of bacteria quantified from the filters compared favorably with the liquid impinger counts. Replicate samples clustered together following principal components analysis of the profiles. We concluded that lipid analysis was comparable with standard sampling procedures where culturable organisms were studied. By enabling rapid quantitative analysis of bioaerosols, lipid analysis will allow characterization of culturable and non-culturable indoor air microbial communities in areas where air quality is in question. Further studies using lipid analysis of non-culturable indoor air pollutants such as endotoxin are currently in progress.

257. PCR for Enhanced Bioaerosol Detection.

L. Stetzenbach, A. Alvarez, M. Buttner, UNLV/HRC, Las Vegas, NV

Monitoring of bioaerosols has traditionally been focused on culture-based methods. However, bacterial cells may be nonculturable due to the stress of aerosolization and aerobiological sampling. Polymerase chain reaction (PCR), used to amplify specific genetic sequences, has been shown to detect airborne microorganisms with greater sensitivity and specificity than culture. Recent laboratory and field experiments were designed to optimize PCR protocols for use in bioaerosol monitoring. The amplification of plasmid target sequence resulted in a detection limit of a single bacterial cell using freeze-thaw and solid-phase cell lysis methods, within 5 and 9 hours, respectively. With a genomic target, the sensitivity of the solid-phase method was 10-fold lower than with the freeze-thaw method. AGI-30 samplers were used to collect environmental bioaerosols before and after inoculation with a target organism to obtain data on sampling stress and interferences due to the presence of non-target DNA. Inhibition to PCR amplification was observed when 10 to 104 CFU/m of environmental organisms were present but successful amplification was obtained with a 10-fold dilution of collection buffer. Sampling stress resulted in an order of magnitude decrease in culturability of cells but no difference in sensitivity of the PCR assay was observed. In a field validation experiment, the presence of airborne E. coli and/or Shigella sp. were detected in outdoor samples using genomic primers. This research verifies that PCR represents a rapid, sensitive alternative to culture-based detection of airborne microorganisms.

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