Indoor Air 1994, 4 239-247 Prinred m Denmark . all rights reserved

Copyr tgh t Munksgaard I 9 9 4

Indoor Air ISSN 0905-6947

Quantification of Muramic Bacterial Peptidoglycan, in

Acid, a Marker for Dust Collected from

Hospital and Home Air-Conditioning Filters using Gas Chromatography - Mass Spectrometry Alvin Fox', Rose Marie T. Rosario]

Abstract The development of standardized non-culture-based ap- proaches capable of assessing microbial contarnination of airborne dust is sorely needed. Direct chemical analysis has previously been successfully used for measuring components unique to Gram-negative bacteria. In the present study, dust from primary filters of hospital air-conditioning intake sys- tems (which filter incoming outdoor and recirculated air) and dust from secondary room filters (filtering primarily indoor air) were analyzed. Dust from home air-condition- ing filters (which also filter outdoor air, with recirculation) were also analyzed. Muramic acid is an aminosugar present in Gram-positive and Gram-negative bacterial cell walls and can serve as a measure of bacterial contamination in dust. Samples were analyzed by gas chromatography-mms spectrometry after hydrolysis and conversion of released sugars (including muramic acid) to alditol acetates. Primary hospital filters contained 26.3 & 10.0 ng of muramic acid/ mg dust while secondary filters contained 5.3 The level of inuramic acid in home air-conditioner dust was 31.71fr 1.3.4 ng/mg. This study of dust collected from air-conditioners demonstrates the feasibility of chemical as- sessment of the microbial contamination of indoor air.

5.4 ng/mg.

KEY WORDS: Muramic acid, Air, Dust, Gas chromatography-mass spectrometry

Manuscript received: 5 July 1993 Accepted for publication: 27 February 1994 A preliminary version of this paper was presented at the Indoor Air '93 Conference in Helsinki in July 1993

' Department of Microbiology and Immunology, University of South Carolina, School of Medicine, Columbia, SC, USA (fax: (803) 733 3192)

Introduction Certain human pathogens (including mycobacteria and legionellae) are likely to be significant causes of infection from indoor air, infecting otherwise healthy individuals (Fraser et al., 1977; Iseman, 1992). However, many opportunistic bacteria found in the indoor environment pose a potential threat only to immunocompromised patients in hospitals. There are various sources of airborne nosocomial infections including dust introduced into the hospi- tal. However, in many buildings in the US, inter- change between outdoor and indoor air is limited and a primary source of interchange is through the filtration system of the air-conditioner. Although there is agreement about the need to reduce the number of bacteria (especially in operating theaters) there are no widely accepted standards for the qual- ity of air inside the hospital due to the difficulty in quantifying bacteria present in airborne dust (Humphreys, 1992; Schaal, 1991).

Gram-positive bacteria are often present in higher concentrations than Gram-negative bacteria in indoor air. However, due to the variety of bacteria present, speciation is complex and rarely exhaustive (Gallup et al., 1993). Considerable diversity is re- ported with Gram-positive cocci (including staphylococci and micrococci), pleomorphic organ- isms (including diphtheroids) and rods (including bacill) all being common (Macher et al., 1991; Har- rison et al., 1992; Gallup et al., 1993; Godish et al., 1993; Maroni. et al, 1993).

In the absence of infection, exposure to endotoxic or immunogenic substances (derived from non-vi- able bacteria remnants) can lead to pulmonary irri- tation and allergic reactions associated with breath- ing contaminated air in the home or workplace

240 Fox & Rosario: Quantification of Muramic Acid in Dust collected from Hospital and Home Air-conditioning Filters

(Malmberg, 1990; Rylander, 1990). This might be related to the sick building phenomenon. The limited introduction of “fresh” air and constant re- circulation creates a fertile breeding ground for bac- teria once air-conditioning systems, humidifiers or water towers become contaminated (Fraser et al., 1977).

Currently, culture is by far the most widely used procedure for assessing the microbial content of in- door air. Unfortunately, selective growth of certain species (an unavoidable limitation of culture tech- niques) may leave the majority of the original bac- terial population undetected. Indeed, in the hostile environment found in air, many microbes are likely to be non-viable. However, toxic cell wall com- ponents (including peptidoglycan and lipopolysac- charide) retain biological activity even after-extreme exposure to physical and chemical stress.

Determination of activation of the clotting cas- cade of Limulus amoebocyte lysate (LAL) by Gram- negative endotoxins (lipopolysaccharides, LPS) has been used as a measure of the levels of bacterial contamination in air (Milton et al., 1992). The LAL does not detect Gram-positive bacteria or peptido- glycan unless present at abnormally high concen- trations. Low concentrations of several other sub- stances will activate the LAL reaction, whereas others will inhibit the test (Kotani et al., 1977; Pear- son et al., 1984). In complex samples such as dust, it is sometimes uncertain whether LPS or some other substances is activating the LAL. A need for pru- dence in interpreting results of endotoxin determi- nations in dust has been suggested (Malmberg, 1990).

An alternative approach to measuring LPS is based on the detection of specific structural com- ponents (chemical markers) of the LPS molecule. Lipid A, the lipid component of LPS that is respon- sible for its endotoxicity, contains a large fraction of 3-hydroxy fatty acids. The presence of 3-hydroxy fatty acids (derived from LPS) in organic dust has been measured by high performance liquid chrom- atography (Morris et al., 1988) and GC-MS (Sones- son et al., 1988; Sonesson et a1.,1990). Although liquid chromatography (LC) and gas chromatogra- phy (GC) both provide excellent separations, con- ventional LC and GC detectors do not provide the exquisite selectivity of mass spectrometry often necessary for trace analysis of complex samples such as dust (Mielniczuk et al., 1992; Mielniczuk et al., 1993).

Certain dust samples contain mainly Gram-nega-

tive bacteria. For example, the majority of bacteria present on cotton dust are Gram-negative, notably Enterobuaer ugglomeruns (Rylander, 1990). High concentrations of Gram-positive bacteria, including Actinomycetes, have been observed in materials that have caused allergic respiratory problems (Malmberg and Rask-Anderson, 1988). In air samples from swine confinement buildings and poultry confinement buildings the vast majority of cultured bacteria are Gram-positive, with most being enterococci (Clark et al., 1983; Jones et al., 1984).

Peptidoglycan, the backbone of the cell wall of both Gram-negative and Gram-positive eubacteria, displays endotoxin-like activity (Fox, 1990) and is likely to pose a severe threat to health in the indoor environment (Sonesson et al., 1988). Other than culture, there have been no assays available for as- sessing the levels of Gram-positive bacteria in air- borne dust or other complex matrices. PG is a single bag shaped, highly cross-linked macromolecule that surrounds the bacterial cell and provides rigidity and thus protection from osmotic lysis. PG is one of the few substances common to almost all eubacteria (whether Gram-positive or Gram-negative) and not present in nonbacterial matter (Fox et al., 1980; Fox et al., 1989; Elmroth et al., 1993; Findlay et al., 1983; Fox and Fox, 1991). PG has a glycan (poly- saccharide) backbone that is a repeating polymer of N-acetylglucosamine and N-acetylmuramic acid. Muramic acid is unique to bacteria and since it does not occur elsewhere in nature it is a definitive marker for bacteria (Fox et al., 1980; Gilbart et al., 1986). Although used to quantify levels of PG in a variety of matrices, there have been few previous measurements of the levels of muramic acid in air- borne dust. The purpose of the present study was to determine levels of muramic acid to monitor the concentrations of Gram-positive and Gram-nega- tive PG in indoor dust from hospitals and homes. A brief report of these observations has appeared elsewhere (Fox and Rosario, 1993) and a follow-up to this study has also recently appeared (Fox, Rosa- rio and Larsson, 1993).

Materials and Methods Intake fan units were located on different floors and on the roof of hospitals studied. Air initially passes through a primary filtration bank and is then recir- culated through another set of filters (secondary fil- ters) located in individual rooms and in hallways.

Fox & Rosario: Quantification of Muramic Acid i n Dust collected from Hospital and Home Air-conditioning Filters 241

Home air-conditioning systems usually contain one filter to remove dust from outdoor air as it enters the building, although there is some recirculation. Dust was collected from primary and secondary fil- ters from hospital as well as home air-conditioning filters by shaking or brushing into collection tubes before freeze drying. Primary dust filters in hospi- tals and in homes filter primarily incoming outdoor and recirculated air whilst secondary room filters filter dust primarily from indoor air. Ages of filters varied and during prolonged use growth may have occurred.

To assess the levels of muramic acid in dust, samples were analyzed using the alditol acetate method (Fox et al., 1989; Fox and Black, 1993). Fifteen to 20 mg of each sample was weighed di- rectly into hydrolysis tubes, 0.6 ml of 2N sulfuric acid was added, and samples were resuspended by sonication in an ice bath. Hydrolysis was then per- formed for 3 h at 100 "C. Methylglucamine (2 pg) was then added as an internal standard along with 1 mg glucose (which acted as a carrier to minimize losses during derivatization and passage through the

GC-MS system). External standards consisted of a mixture of 1 mg of glucose, 500 ng of muramic acid and 2 pg of methylglucamine. Samples were then neutralized by mixing with 1.5 ml N,N-dioctylme- thylamine: chloroform (50: 50 vol/vol). The aque- ous phase was passed through a C-18 column (Ana- lytichem, .Harbor City, CA) to remove hydrophobic contaminants and reduced with 5 mg sodium boro- deuteride. T o remove borate (generated from so- dium borodeuteride), which otherwise inhibits the acylation reaction, multiple doses of methanol-acet- ic acid (200:l vol/vol) were added to the samples while evaporating under nitrogen using a custom- built automated evaporator. The samples were then dried under vacuum. The alditols were acylated with acetic anhydride at 100 "C overnight. Acetic anhydride was decomposed with 0.75 ml of water. One ml of chloroform was added and after mixing, the aqueous phase was discarded. 0.8 ml of concen- trated ammonium hydroxide: water (80 : 20 volume/ volume) was added. The mixture was passed through a magnesium sulfate column (Chem Elut columns, Analytichem) and the chloroform phase

3 0 0 0 0 0

2 0 0 0 0 0

100000

W 0

6 0 8 0 100 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0

8 o o o o ~ I 100

B

6 0 0 0 0

40000

2 0 0 0 0

0 444 437J

6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 180 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 400 4 2 0 4 4 0 4 6 0

m/z Fig. 1 Moss spectra of alditol acetates of a muramic acid standard prepared with (A) acetic anhydride (B) deuterated acetic anhydride

242 Fox & Rosario: Quantification of Muramic Acid in Dust collected from Hospital and Home Air-conditioning Filters

collected. The samples were evaporated to dryness and resuspended in 30 pl of chloroform.

GC-MS analyses were carried out with a mass selective detector (model 5970, Hewlett-Packard Co., Avondale, PA) interfaced to a GC (model 5890, Hewlett-Packard Co.) equipped with an automated sample injector (model 7673A, Hewlett-Packard Co.) and an SP-2330 fused-silica capillary column (Supelco, Bellefonte, PA). Selected ion monitoring (SIM) in low resolution mode was performed with ionization at 70 eV. Ions m/z 168, m/z 404 and m/ z 446 for muramic acid and m/z 170 for methylglu- carnine were monitored. In some experiments con- firmation of sugar identities were made by substi- tuting deuterated acetic anhydride for normal acetic anhydride in preparation of alditol acetates. Pre- dicted shifts in m/z for characteristic ions were then monitored.

Results In the present study muramic acid was measured to assess the levels of Gram-negative and Gram- positive bacterial PG. The levels of this compo- nent in some of the samples were low and se- lected ion monitoring (SIM) was essential. SIM

60000

50000

40000 55

dramatically increases the sensitivity of detection by focussing on characteristic ions for the com- pound of interest. Although SIM is highly selec- tive, chemical identification is not as absolute as when a full mass spectrum is obtained by total ion monitoring.

The alditol acetate of muramic acid (when the C- 1 aldehyde is labelled with deuterium) has a molecu- lar weight of 464. Loss of water allows the formation of an amide bond between the C-2 nitrogen and the carboxyl group of the lactyl moiety producing muramicitol pentaacetate lactam (molecular weight 446). The other high molecular weight ion, m/z 404, is generated by loss of ketene (m/z 42). The most prominent low molecular weight ion is m/z 168. The mass spectrum of the alditol acetate of standard muramic acid prepared using acetic anhy- dride is shown in Figure 1A. Many characteristic ions are clearly observed in the mass spectrum of muramic acid isolated from dust removed from an air-conditioning filter when compared to the pure compound (see Figure 2). However, appreciable background is clearly present in the dust sample. For example, in the standard m/z 168 has the highest abundance, whilst in the dust sample m/z 115 dominates the spectrum.

A

30000

20000

w 10000

El

V 5 0

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460

3 97

8 0 0 0 0

60000

40000

20000

0 6

1

86

80 100 :

B 3

130

461

0 140 160 180 200 220 240 260 280 300 320 340 3 i O 380 400 420 440 460

mlz Fig. 2 Mass spectra of alditol acetates of muramic acid from dust sample prepared with (A1 acetic anhydride (BI deuterated acetic anhydride

Fox 8. Rosario: Quantification of Muramic Acid in Dust collected from Hospital and Home Air-conditioning Filters 243

Thus in order to detect muramic acid, SIM was essential for maximal selectivity and specificity. Three ions characteristic of muramic acid (m/z 168, 404 and 446) were monitored. The relative abun- dances of these selected ions in dust samples were similar to those for standard muramic acid (see Table 1). In order to provide additional codir- mation of the presence of muramic acid in dust, deuterated acetic anhydride was used in place of non-deuterated acetic anhydride. Since each acetate group contains 3 deuteriums and there are five ace- tate groups in muramicitol pentaacetate, it can be predicted that ion m/z 446 will be replaced by m/ z 461. Since ion m/z 404 is generated by loss of ketene (which contains 2 deuteriums), it can also be predicted that this ion will be replaced by m/z 417. The predicted results indeed occurred as shown in the mass spectra of a standard and dust sample deri- vatized with non-deuterated acetic anhydride (Fig- ures 1A and 2A) which should be compared with samples prepared with deuterated acetic anhydride (Figures 1B and 2B).

In order to more readily observe the predicted mass shifts, four ions (m/z 404, 446, 417 and 461) were used to monitor muramic acid in samples after derivatization with acetic anhydride or deuterated acetic anhydride. Ions m/z 404 and 446 (but not m/z 417 and 461) would be predicted to detect mur- amic acid in samples after reaction with acetic anhy- dride. Ions m/z 417 and 461 (but not m/z 404 and 446) would be predicted to detect muramic acid in samples after reaction with deuterated acetic anhy- dride. The predicted mass shifts are readily ob- served by comparing both standards (see Figure 3) and dust samples (see Figure 4).

Dust collected from six home filters was found to contain 31.72 13.4 ng of muramic acid per mg of dry dust. Dust from five primary hospital filters was analyzed and found to contain 26.3 f 10.0 ng of muramic acid per mg dust. Dust from ten secondary hospital filters was analyzed and found to contain 5.3 k 5.4 ng of muramic acid per mg of dust. Figure

Table 1 Rela:ive abundances of three ions monitored at the re tention time for muramic acid in dust samples and standards

miz 4041 mlz 168 mlz 4461 mlz 168

Standard 0.539 0.140 0.543 0.153

Dust sample 1 0.545 0.160 0.529 0.161

Dust sample 2 0.558 0.164 0.597 0.140

5 shows chromatograms illustrating muramic acid levels present in dust collected from hospital pri- mary and secondary filters. By observing the ratio of muramic acid to the internal standard methylglu-

90000

80000

70000

60000

Iethylqluosnine

Iuuramlc Acid

0 . . . . . . . . . . . . . . . . . . . . . . , . . . . , . i m e -= ia:oo i o : o o 22:oo 14loo 16:o.a 28.00 30.00

Ion 4 0 4

. . . . . . . . . . . . I . . . . . . . . . . . . I . lo0 20100 22100 24 .00 26lOO 1 8 : O O 30.00

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0 18% 20100 22100 - 24.00 26.00 28.00 30.00

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n i . . . n . . . . . . . . I . . . . , . . . . , . . . . , . . . . imc -> 1 0 . 0 0 20.00 22.00 24.00 26.00 2 8 . 0 0 30.00

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0. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.00 20.00 22.00 2 4 . 0 0 26.00 28.00 3 0 . 0 0

Ion 417

E f O b ' ' 2 O : O O ' ' 22:OO ' ' ;<lob ' ' i610O ' ' 2 8 f O O ' ' JQ10b

Ion 461

l8!00 1O:OO 22100 1 4 . 0 0 26.00 2 8 . 0 0 30.00

o ] A , , . . , , 1 , , * . . , , , , , , , , , , . ,

Fig. 3 Selected ion chromotograms of standards prepared with (A) acetic anhydride and (6) deuterated acetic anhydride

244 Fox 8, Rosario: Quantification of Muramic Acid in Dust collected from Hospital and Home Air-conditioning filters

A 250000' n.thylplucamIn*

Cluco.anin. nuranic AcId ~ 0 0 0 0 0 ~

150000- I

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5 0 0 0 0 ~

0 - . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . F l u -s lsloo 20:oo 22:oo 24:OO 26:OO 2.100 ao.oo

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m l o o zo!oo 22l00 14100 is100 m l o o ao.oo

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mloo 20100 22.00 24.00 26.00 28.00 ao.oo

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0 . . . . . . . . . . . . . . . . . . . . . . 8 . . . I

ieloo d o o 22:oo 2rloo 26100 ~ 8 . 0 0 ao.00 I 1 Ion 446

Ion 4 6 1

' ' ' l 8 ! 0 0 ' ' 2'0!00 ' ' 2'2!00 ' ' 2 4 ! & ' 26100 28100 lO!OO

Fig. 4 Selected ion chromatograms of dust hydrolysate pre- pared with IA) normal acetic anhydride IB) deuterated acetic an- hydride. An additional glucosamine peak is observed in the dust samples but not in standards

camine it is clearly seen that muramic acid is present at much higher levels in primary filters. Table 2 documents the levels of muramic acid in the three different environments.

Discussion There is a real need for a means of monitoring bac- terial concentrations in airborne dust present in hos- pitals as a potential source for opportunistic infec- tion. In a wider sense it is becoming recognized that airborne bacterial allergens /endotoxins may be a significant cause of respiratory symptoms after en- vironmental exposure in homes and industrial buildings.

Measurements of the levels of viable bacteria can provide false estimates of the content of bac- terial components in dust. There has alternatively been a major focus on the content of bacterial endotoxins (LPS) derived from airborne Gram- negative bacteria, and numerous studies have suggested a correlation between the level of

40000

3 0 0 0 0

25000

20000

15000

10000

A il Glucosamine

1 Methylglucamine B

0 , . , I '

TIME 2 5 . 0 0 3 0 . 0 0 3 5 . 0 0 40.00

Fig. 5 Selected ion chromatograms of alditol acetates of (A) primary dust hydrolysate (B) secondary dust hydrolysate. Note that the ratio of muramic acid to the internal standard lmethylglu- caminel i s much higher in the primary dust sample

Fox & Rosario: Quantification of Muramic Acid in Dust collected from Hospital and Home Air-conditioning Filters 245

Table 2 ConceC3tration of muramic acid in dust Ing/mg) collected from home and hospital air-conditioners (values include dupli- cates for each sample analyzed)

Home Primary Secondary

10, 20 28,54 20, 19 50, 50 25, 23 11, 6 20, 20 21, 20 3, 4 40, 40 22, 24 2, 2 40, 40 23, 23 4, -

30, 20 3, 3 3, 4 3, 2 2, 2 3, 5

Gram-negative bacterial endotoxin in indoor air and pulmonary disorders (Attwood et al., 1987; Castellan et al., 1984; Cinkotai et al., 1977, Olen- chock et al., 1982). It has been suggested that airborne PG - a structure capable of causing chronic inflammatory reactions - may also play a role in pulmonary dysfunction resulting from ex- posure to contaminated air (Sonesson et al, 1988).

As traditional methods for measuring LPS in air samples are unsatisfactory, there is a real need for an alternative standard approach for charac- terizing the microbial components of organic dust. With chemical methods, the total bacterial load is measured with no discrimination between live and dead organisms. Certain hydroxy fatty acids have been used as chemical markers to ac- curately assess the levels of endotoxin in contami- nated air. There have been few studies on the levels of PG in airborne dust (Sonesson et al., 1988; Fox, Rosario and Larsson, 1993); however, in certain indoor air environments (e.g. poultry and swine farms), where Gram-positive bacteria may greatly outnumber Gram-negative bacteria, it would be useful to have a measure of PG (Clark et al., 1983; Sonesson et al., 1990). A marker widely distributed among bacterial spe- cies, but absent elsewhere in nature, is muramic acid found in the bacterial cell wall PG. This aminosugar can be used to measure the levels of bacterial PG without microbiological culture.

In the present study the levels of muramic acid were measured in dust from air-conditioning filters of homes and hospitals. The purpose was primarily to develop the analytical method and demonstrate feasibility of the approach. Conclusions about in- door air quality will await results of air monitoring. In most homes the indoor environment is directly

connected, via a single air-conditioning filter, to the outside environment. Because of concern about op- portunistic bacterial infections, many hospital fil- tration systems are more elaborate and designed to remove as much dust and associated bacterial par- ticles as possible. Dust from primary air-condition- ing filters (designed to filter incoming air from the outdoor environment and from recirculation) and secondary filters from individual rooms (removing dust from indoor air) was analyzed. Home air-con- ditioning filters collect dust in an analogous fashion to hospital primary filters.

In order to confirm identity of muramic acid, deuterated acetic anhydride was used in place of acetic anhydride during derivatization; the molecu- lar species were shown to alter in the predicted fashion on MS. The mean levels of muramic acid in dust collected from hospital primary filters was 26.3 10.0 ng per mg/dry dust. The muramic acid levels in dust collected from secondary filters was 5.3 k 5.4 ng/mg. These results suggest that there is a significant reduction of the bacterial content of dust in the indoor hospital environment resulting from air filtration. Levels of muramic acid in dust collected from homes (31.7 k 13.4 ng/mg) were not significantly different from the levels of muramic acid from hospital primary filters. This is not sur- prising since hospital primary filters and home fil- ters both primarily remove dust from outdoor air as it enters the building.

It is possible that dust collected from secondary filters in hospitals is not as conducive to growth of bacteria as dust collected from primary hospital fil- ters or home filters. This may reflect a decrease in nutrient content as soil and plant matter in dust is replaced by lint from clothing, bandages and equip- ment.

In conclusion, bacteria and bacterial debris can be detected in airborne dust without prior culture by measuring levels of a chemical marker for bac- terial peptidoglycan. These results demonstrate the feasibility for chemical measurement as a means of monitoring the content of bacteria and their con- stituents in the air of hospitals, homes and work- places.

Acknowledgements This study was supported by the Center for Indoor Air Research (92-03A) and Army Research Office (DAAL03-92- 1255).

246 Fox 8, Rosorio: Quantification of Muromic Acid in Dust collected from Hospital and Home Air-conditioning Filters

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