An integrated procedure is presented whereby gas chromatography-ion trap mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 10 to 18 carbon atoms), gram-positive bacteria (branched-chain fatty acids with 15 and 17 carbon atoms), bacterial peptidoglycan (muramic acid), and fungal biomass (ergosterol) in samples of settled house dust. A hydrolysate of 13 C-labeled cyanobacterial cells is used as an internal standard for the first three markers. These analyses require two dust samples, one for 3-OH fatty acids, branched-chain fatty acids, and muramic acid and another for ergosterol. The method may be used to characterize microbial communities in environmental samples.Inhalation of airborne microorganisms in indoor environments has been associated with the development of respiratory disorders. Substances such as endotoxins (lipopolysaccharides [LPS]) (14,17,18,28), peptidoglycan (9, 12), and various fungal components and products (3,6,21) are among the suspected causative agents. Culture-based methods are suitable for detection of culturable infectious agents and allow species identification; however, it is widely agreed that only a small fraction (0.1 to 10%) of the total microbial flora in an indoor environment is currently culturable (29). Direct microscopy is at best semiquantitative. Although the Limulus amebocyte lysate test is extremely sensitive to endotoxin and can detect glucans, it measures bioactivity rather than absolute amounts and its reproducibility and specificity have been questioned (5). Nucleic acid-based methods including PCR are very specific, and by using broad-range (universal) probes and primers sets based on 16S rDNA (20) and 18S rRNA (31), most bacteria and fungi present in a sample can be identified. To date, such universal probes and primers have not been used to characterize the microbiology of indoor environments; rather, PCR has been used to detect specific fungi and bacteria in such environments (4,11,22).In our laboratory we have developed and applied gas chromatography-mass spectrometry (GC-MS) methods to determine biomarker molecules in complex matrices including organic dust. Microorganisms contain unique compounds not found elsewhere in nature, which can be used as chemical markers of larger, bioactive structures (7). Endotoxins (LPS) are major constituents of the outer membrane of gram-negative bacteria. A backbone of lipid A, the toxic component of the LPS molecule, carries in general 4 mol of unique 3-hydroxy fatty acids (3-OH FAs) (23-26). Certain branched-chain FAs are found in most gram-positive bacteria (13,30). Muramic acid is a unique marker of peptidoglycan (2,8,9,15), which is the cross-linked macromolecular structure responsible for the rigidity of bacterial cell walls and is sometimes referred to as "gram-positive bacterial endotoxin" (27). Ergosterol is a common fungal membrane lipid that is widely used as a marker of fungal biomass, although the concentration depends on the species and ...

Microbial exposure has been indicated as significant in the development of asthma and allergy among children. The aim of the study was to test whether microbial exposure and allergens in the school environment are associated with asthmatic symptoms in pupils. Data on asthmatic symptoms and respiratory infections were collected through a questionnaire survey among 1993 pupils aged 11-15 yr in 10 randomly selected schools in Taiyuan, China. Settled dust in classrooms was analysed using tandem gas chromatography-mass spectrometry for 3-hydroxy fatty acids, marker of lipopolysaccharide (LPS) from endotoxin, muramic acid (MuA), marker of bacteria and ergosterol (Erg) for fungi, quantifying both culturable and non-culturable microbes. A total of 29.8% reported daytime attacks of breathlessness, 8.4% wheeze and 1.2% had doctor's diagnosed asthma. Generally, MuA was negatively associated with wheeze and daytime attacks of breathlessness, the latter of which was negatively associated with Erg to a weaker extent. Total concentration of LPS was positively associated with daytime attacks of breathlessness, but shorter lengths of LPS, C10, C12 and C14 LPS were negatively associated with either wheezing or daytime attacks of breathlessness. For MuA and C10 and C12 of LPS, the associations were independent of airborne allergens and classroom crowdedness, and even independent of the other two microbial markers for MuA. Microbial exposure indicated by certain chemical markers (e.g. MuA) could be protective for asthmatic symptoms, but for LPS (endotoxin), the picture is more complex, varying by different lengths of fatty acids of LPS.

Our group previously demonstrated that carbon dioxide (CO2) levels in heavily occupied schools correlate with the levels of airborne bacterial markers. Since CO2 is derived from the room occupants, it was hypothesized that in schools, bacterial markers may be primarily increased in indoor air because of the presence of children; directly from skin microflora or indirectly, by stirring up dust from carpets and other sources. The purpose of this project was to test the hypothesis. Muramic acid (Mur) is found in almost all bacteria whereas 3-hydroxy fatty acids (3-OH FAs) are found only in Gram-negative bacteria. Thus Mur and 3-OH FA serve as markers to assess bacterial levels in indoor air (pmol m(-3)). In our previous school studies, airborne dust was collected only from occupied rooms. However, in the present study, additional dust samples were collected from the same rooms each weekend when unoccupied. Samples were also collected from outside air. The levels of dust, Mur and C10:0, C12:0, C14:0, and C16:0 3-OH FAs were each much higher (range 5-50 fold) in occupied rooms than in unoccupied school rooms. Levels in outdoor air were much lower than that of indoor air from occupied classrooms and higher than the levels in the same rooms when unoccupied. The mean CO2 concentrations were around 420 parts per million (ppm) in unoccupied rooms and outside air; and they ranged from 1017 to 1736 ppm in occupied rooms, regularly exceeding 800-1000 ppm, which are the maximum levels indicative of adequate indoor ventilation. This indicates that the children were responsible for the increased levels of bacterial markers. However, the concentration of Mur in dust was also 6 fold higher in occupied rooms (115.5 versus 18.2 pmole mg(-1)). This further suggests that airborne dust present in occupied and unoccupied rooms is quite distinct. In conclusion in unoccupied rooms, the dust was of environmental origin but the children were the primary source in occupied rooms.

The authors assessed determinants of ergosterol, 3-OH fatty acids (FAs), and viable microbes in vacuum cleaner dust, and investigated the association between these microbial markers and childhood asthma. The authors studied the homes of 36 children who were new cases of childhood asthma and the homes of 36 controls. Home characteristics explained 34% to 44% of the variation in levels of different microbial groups. Determinants of 3-OH FAs were a lower level of cleanliness, having a fireplace, having livestock, and moisture damage; determinants of viable bacteria were the level of home repair needed and the material used in the building frame of the home. Ergosterol was associated with the presence of livestock and the practice of cleaning rugs outside; viable fungi was associated with the material used in the building frame, visible mold, and the practice of cleaning rugs outside. Exposure to mesophilic actinomycetes was nonsignificantly associated with risk of asthma. The authors concluded that the variation of microbial levels in dust could be explained relatively well by home characteristics, and suggested that exposure to mesophilic actinomycetes may increase the risk of new asthma.

Exposure to environmental tobacco smoke (ETS) is an important worldwide public health issue. The present study demonstrates that cigarette smoke can be a major source of endotoxin (lipopolysaccharide, LPS) in indoor environments. Gas-chromatography/mass-spectrometry was used to determine 3-hydroxy fatty acids as markers of endotoxin in air-borne house dust in homes of smokers and non-smokers. Air concentrations of endotoxin were 4-63 times higher in rooms of smoking students than in identical rooms of non-smoking students. The fact that cigarette smoke contains large amounts of endotoxin may partly explain the high prevalence of respiratory disorders among smokers and may also draw attention to a hitherto neglected risk factor of ETS.