Potentially Pathogenic, Slow-Growing Mycobacteria

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Journal of Occupational and Environmental Hygiene, 1: 1–6

ISSN: 1545-9624 print / 1545-9632 online

Copyright c 2004 JOEH, LLC

DOI: 10.1080/15459620490250008

Potentially Pathogenic, Slow-Growing Mycobacteria

Released into Workplace Air During the Remediation

of Buildings

Sirpa Rautiala,1 Eila Torvinen,2 Pirjo Torkko,2,3 Sini Suomalainen,4

Aino Nevalainen,2 Pentti Kalliokoski,5 and Marja-Leena Katila3

1Kuopio Regional Institute of Occupational Health, Kuopio, Finland

2National Public Health Institute, Kuopio, Finland

3Kuopio University Hospital, Kuopio, Finland

4University of Helsinki, Helsinki, Finland

5University of Kuopio, Kuopio, Finland

Construction workers' exposure to airborne viable mycobacteria

was studied during the remediation of three moldy

and two nonmoldy buildings. Furthermore, the concentrations

of airborne fungal and actinobacterial spores were determined.

The samples for the microbial analyses were collected using a

six-stage impactor and an all-glass impinger sampler, and by

filter sampling. Specific mycobacteria media and nonselective

media were used for the cultures. The samples were cultured

for the total numbers of rapidly growing and slow-growing

mycobacteria, and the isolates obtained were identified to the

genus or species level. Mycobacteria were recovered from the

air during the remediation of two of the moldy buildings and

one nondamaged building. Concentrations of mycobacteria

up to 160 cfu/m3 were detected. A total of 43 mycobacterial

isolates was recovered. Most of the isolates were slowgrowers,

only two rapid-growing strains being detected. The 38

identified isolates belonged to potentially pathogenic species,

including Mycobacterium avium complex, M. scrofulaceum,

and M.fortuitum, and to saprophytic species, including M.

nonchromogenicum and M. terrae. Mycobacteria were the

most often detected in samples taken with a six-stage impactor.

They were found in buildings with both high and low concentrations

of fungi. In conclusion, mycobacteria, both potentially

pathogenic and saprophytic species, may be released into the

indoor air during the remediation of buildings.

Keywords airborne mycobacteria, construction work, indoor air

Address correspondence to: Sirpa Rautiala, Kuopio Regional

Institute of Occupational Health, P.O. Box 93, 7071 Kuopio, Finland;

e-mail: sirpa.rautiala@....

Constructionworkers have been found to be exposed

to high concentrations of fungal and actinobacterial

spores during the remediation of moldy

structures.(1,2) Such exposure has also been found

to be associated with an increased prevalence of respiratory

symptoms, including dry cough, runny nose, eye irritation,

and hoarseness.(3) It has been suggested that actinobacterial

and fungal spores released from damaged building materials

would cause these kinds of health effects. However, there

are several other biologically active microorganisms (e.g.,

bacteria)(4,5) present simultaneously although they have not

been well characterized. Currently, the role of various

microorganisms

as possible causal agents of the health effects remains

obscure.

Mycobacteria are acid-fast, slow-growing Gram-positive

bacteria. The most harmful mycobacteria for man are the

human tuberculosis and leprosy bacilli. Environmental mycobacteria,

which are also called atypical mycobacteria, or

mycobacteria other than Mycobacterium (M.) tuberculosis,

are heterotrophic species that take part in the decomposition

of organic matter. Environmental mycobacteria occur in

soil and natural waters,(6,7) but also in man-made water

environments.(8,9)

Environmental mycobacteria can be divided into two main

groups on the basis of their growth rate. The rapid-growing

mycobacteria are seen as visible colonies grown from a dilute

inoculum in less than 7 days, while the slow-growing mycobacteria

usually need several weeks before visible colonies

appear.(10) In general, slow-growers are usually potentially

pathogenic to humans and animals, while many of the rapidgrowing

mycobacteria are regarded as nonpathogenic.

Infections caused by environmental mycobacteria are becoming

increasingly common in the United States and other

parts of the world.(11,12) There is no evidence of person-toperson

transmission, and the environment is regarded as the primary

source of infections. Slow-growing mycobacteria (e.g.,

M. avium, M. intracellulare, and M. scrofulaceum) can cause

Journal of Occupational and Environmental Hygiene January 2004 1

chronic pulmonary diseases resembling tuberculosis, cervical

lymphadenitis, and skin and other soft-tissue infections.(11,12)

Mycobacteria are also effective stimulators of the immune

system (i.e., they are capable of stimulating macrophages

to produce inflammatory mediators such as cytokines).(13,14)

Furthermore, aerosolized mycobacteria have been associated

with hypersensitivity pneumonitis among metal workers.(15)

Evidently, the sources of environmental mycobacteria are still

insufficiently known, as is their importance as a causal agent

of occupational diseases.

The slow growth rate of mycobacteria makes it difficult to

isolate them from samples rich in other microorganisms. Incubation

times of 6 weeks to 5 months are mostly needed for the

detection of slow-growing mycobacteria. Decontamination is

an essential part of their culturing, necessary for the destruction

of more rapid-growing flora. If too harsh, decontamination

becomes detrimental, not only to contaminating flora, but also

to mycobacteria, and, if too mild, it may allow the growth of

fast-growing contaminating microorganisms, which prevent

the growth of mycobacteria. In addition, selective media are

needed for the isolation of slow-growing mycobacteria.

Environmental mycobacteria have only recently been taken

into account in the study of moldy indoor environments and

assessments of their adverse health effects. Rapid-growing

mycobacteria have been isolated from the indoor wall of

a children's day care center, where workers had reported

work-related upper respiratory symptoms.

(5,16) No previous

results have been reported on the occurrence of slow-growing

mycobacteria in indoor air. To identify their possible role

as exposing agents via inhalation, we examined whether

mycobacteria can be isolated from air during the remediation of

moldy buildings and buildings without detectable mold damage.

In parallel with mycobacterial sampling, the occurrence

of fungi and actinobacteria were also studied to determine

whether there is any association between concentrations of

mycobacteria and other microorganisms.

MATERIALS AND METHODS

Sampling Strategy

Viable airborne mycobacteria, fungi, and actinobacteria

were sampled in five buildings during the dismantling of

the structures (Table I). Three of the studied buildings had

suffered from water damage and had visible microbial growth

on the surfaces of the structures. In Buildings 3 and 4, the

water damage had occurred recently, but in Building 5 the

water damage was older, having occurred years earlier. In

these damaged buildings, air samples were taken during the

dismantling of moldy materials. The two reference buildings

had no knownhistory ofwater damage and had no signs of mold

growth on their surfaces, but they were undergoing another type

of remediation. Air samples were taken during the dismantling

of nondamaged wall materials in these buildings. All the air

samples were taken as close to the remediation process as

possible, generally within 5 meters of the worker.

TABLE I. The Buildings Studied

Reason for RemediationWork

Building Mold Growth Examined

1. Nonmoldy — removal of the top layer

of a concrete wall by

grinding

2. Nonmoldy — dismantling of a wooden

panel wall

3. Moldy water leaks dismantling of a wall

made of gypsum board

4. Moldy leaks from

pipes

dismantling of both a

wall made of gypsum

board and wooden

floor

5. Moldy, old

water damage

leaks from

pipes

dismantling of a wooden

floor

One set of three parallel samples for mycobacteria was

collected using a six-stage impactor, an all-glass impinger,

and by filter sampling at each site. Air samples for fungi

and actinobacteria were only taken with impactors, one to

four samples being taken during the remediation work in each

building. In all, 15 air samples for mycobacteria and 42 samples

for fungi and actinobacteria were taken. Furthermore, one

outdoor air sample was taken as a control at each sampling

site using impactor, impinger, and filter sampling.

Isolation of Microorganisms

Impactor Samples

Impactor samples were taken with six-stage cascade impactors(

17) (Model 10-800, Andersen Samplers, Inc., Atlanta,

Ga.) calibrated at a flow rate of 28.3 L/min. The mycobacterial

samples were collected onto petri dishes containing

Mycobacteria 7H11 agar (Difco Laboratories, Detroit, Mich.)

supplemented with OADC enrichment (100 mL/L), malachite

green (25 mg/L), and cycloheximide (500 mg/L).(18) Dichloran

glycerol agar (DG18)(19) was used for xerophilic fungi, and 2%

malt extract agar (M2)(20) was applied for hydrophilic fungi.

For actinobacteria, tryptone-yeast-glucose agar (TYG)(21) was

used for the collection. The collection time varied between

5 and 15 min for all the samples.

After the collection, the mycobacterial plates were sealed

with parafilm, packed in a plastic bag and incubated at

30 & #9702;C for 6 months. If a plate became visibly contaminated,

which was considered a sign that other microorganisms were

growing, it was eliminated from the incubation. The plates

for fungi and actinobacteria were incubated at 25 & #9702;C for 7

days. In the TYG media, only actinobacteria were counted.

The concentrations of microorganisms were calculated using

a positive hole correction method(17) and expressed as colony

forming units/m3 (cfu/m3).

2 Journal of Occupational and Environmental Hygiene January 2004

All-Glass Impinger Samples (AGI-30)

An AGI-30 impinger was filled with deionized water

(40.5 mL) and sterilized. The flow rate used varied from 10.2

to 12.3 L/min and the sampling times from 30 to 125 min.

The volume of the AGI-30 impinger solution was aseptically

measured after the sampling. Ten milliliters of the solution

was fixed in 2% (w/v) formalin. The rest of the solution was

centrifuged (8600 g, 4 & #9702;C, 15 min, Sorvall RC-5B, E.I. du Pont

Nemours and Co., Wilmington, Del.) and the sediment was

suspended in 5 mL of sterile deionized water. This suspension

was decontaminated with NaOH (final concentration 0.5 M)

for 15 min.

After centrifugation in the same manner as used for the

aforementioned solution, the sediment was neutralized by

adding 30 mL of sterile deionized water, and the sample was

centrifuged, as for the aforementioned solution. The sediment

was resuspended in 400 & #956;L of sterile deionized water, and

50 & #956;L was inoculated onto two parallel slopes of each of

the following growth media: (a) egg medium supplemented

with glycerol, pH 6.5; ( egg medium supplemented with

Na-pyruvate, pH 6.5; © egg medium supplemented with

glycerol, pH 5.5; and, (d) egg medium supplemented with

Na-pyruvate, pH 5.5.(22,23) All the media contained cycloheximide

(500 mg/L). The samples were incubated as described

earlier.

Filter Samples

Filter samples were collected on polycarbonate membrane

filters (diameter 37 mm, pore size 0.4 & #956;m; Nuclepore Corp.

Cambridge, Mass.) with a flow rate of 2 L/min. The sampling

time varied between 30 and 120 min depending on the length

of the remediation work. The microorganisms were eluated

from the filter to 5 mL of sterile peptone water and shaken on a

laboratory shaker for 15 min. After the eluation, the suspension

was decontaminated and cultivated in the same manner as the

described AGI-30 impinger samples.

Identification of Mycobacteria

Each colony type appearing on the growth medium was

examined for acid fastness by Ziehl-Neelsen staining. Acidfast

isolates were subcultured and identified using an identification

scheme for mycobacteria based on the gas liquid

chromatographic (GLC) analysis of cellular fatty acids and

alcohols and mycolic acid cleavage products as described

earlier in detail.(24)

In addition to the analysis for the growth and biochemical

characteristics, the isolates were analyzed using commercial

DNA probes for M. avium complex (AccuProbe; GenProbe,

Inc., San Diego, Calif.).(24) The isolates positive with the

M. avium complex probe were tested further using speciesspecific

probes for M. avium and M. intracellulare. The isolates

were assigned as MAC X if they were positive with the

M. avium complex probe but negative with the M. avium or

M. intracellulare specific probe.(25) The AccuProbe complex

negative isolates that had a GLC profile typical of MAC

and gave a negative result in the Tween 80 hydrolysis test

were sequenced for partial 16S rDNA as described earlier in

detail.(24)

RESULTS

Mycobacteria were recovered from two of the three moldy

buildings and from one of the two nonmoldy buildings

(Table II). The total isolation frequency of the sites studied

was 60% and that of the samples was 33%. The concentrations

of mycobacteria determined for the positive sites varied from

5 to 160 cfu/m3 during the dismantling of the structures. None

of the outdoor air samples yielded mycobacteria in culture

(detection limits 2–68 cfu/m3). Mycobacteria were the most

often detected when sampled with a six-stage impactor. The

highest numbers of viable mycobacteria were detected in the

moldy building where the water damage had occurred years

earlier.

A total of 43 mycobacterial isolates were recovered for

the identification tests. Most of the isolates (95%) were

slow-growers. Only two rapid-growing isolates were detected.

Twenty-four isolates were identified byGLCfatty acid analysis

combined with biochemical testing as M. avium complex.

Among them, 11 hybridized with commercial probes. Three

were identified as M. avium, and eight were MAC X complex

(Table II). The 13 isolates with a typical GLC profile of M.

avium complex, but negative with the M. avium complex probe,

were also analyzed by 16SrRNAgene sequencing. One of them

was identified as M. scrofulaceum, and 12 were grouped very

closely with M. intracellulare. Compared with the sequence of

the type strain of M. intracellulare, the isolates in this group

were found to have three to five deviations within the 1000

bases analyzed, covering both hypervariable regions of the 16S

rRNA gene.

The other slow-growing isolates were identified as

M. terrae or M. nonchromogenicum. Four isolates remained

without a precise identification. One of the isolated rapidgrowing

mycobacteria represented a potentially pathogenic

species, M. fortuitum; the other remained unidentified.

The concentrations of fungal spores in air varied between

102 and 104 cfu/m3 during the remediation of the moldy buildings

and between 102 and 103 cfu/m3 in the nonmoldy buildings

(Table II). In the outdoor air samples, the concentrations of

fungal spores varied between 101 and 102 cfu/m3, depending

on the time of year of the sampling. No actinobacteria were

found in the indoor or outdoor samples.

DISCUSSION

To our knowledge, this is the first report of slow-growing

environmental mycobacteria isolated from indoor air samples.

Slow-growing mycobacteria have earlier been isolated

from air only when sampled above river waters.(18,26) In

the river water studies, mycobacteria of the M. aviumintracellulare-

scrofulaceum (MAIS) complex were recovered

in 0% to 75% of the air samples collected, depending on the

Journal of Occupational and Environmental Hygiene January 2004 3

TABLE II. Concentrations of Microorganisms (cfu/m3) and Species of

Mycobacteria Isolated During the Dismantling of Structures

MycobacteriaA FungiB

Six-Stage Impactor AGI-30 Impinger Filter Sampling

Six-Stage Impactor

Building cfu/m3 Species cfu/m3 Species cfu/m3 Species cfu/m3 (range)

1. Nonmoldy b.d.C b.d.D b.d.E 130–1200

2. Nonmoldy 5 unidentified, rapid-growing

chromogenic species (n = 1)

b.d.D b.d.E 190–600

3. Moldy b.d.C b.d.D b.d.E 120–85,000

4. Moldy 7 unidentified, slow-growing

nonchromogenic species

(n = 1)

b.d.D b.d.E 5020–16,000

5. Moldy, old

water damage

150 M. avium (n = 3)

MAC X (n = 6)

M. intracellulare-like (n = 11)

M. scrofulaceum (n = 1)

M. terrae (n = 7)

M. fortuitum (n = 1)

isolates belonging to

unidentified, slow-growing,

nonchromogenic species

(n = 3)

29 MAC X (n = 1)

M. intracellalare-like (n = 1)

M. terrae (n = 1)

160 MAC X (n = 1)

M. terrae (n = 4)

M. nonchromogenicum

(n = 1)

8200–25,000

Outdoor b.d.C b.d.D b.d.E 2–980

Notes: M. = Mycobacterium; b.d. = below detection limit.

AEach result represents one sample.

BN = number of samples (1–4 per building site) total N = 28.

C Detection limit = 2 cfu/m3 for 15 min sampling and 7 cfu/m3 for 5

min sampling.

DDetection limit = 5–19 cfu/m3.

E Detection limit = 18–68 cfu/m3.

4

environment and season studied. The maximum concentrations

of MAIS complex in the aerosol samples, collected using

a six-stage impactor, were 70 cfu/m3.(26) In the indoor air

of other occupational environments, such as a metalworking

industrial facility, rapid-growing environmental mycobacteria

have recently been detected.(15,27)

M. avium, capable of causing tuberculosis-like infections in

humans and animals, was recovered from indoor air during the

dismantling of the structures. Nonpathogenic mycobacterial

species were also isolated. Although the nonpathogenic species

are unlikely to cause respiratory infections in healthy individuals,

they are known as strong immunostimulators. Some of the

isolates recovered in our study have been shown to be capable

of activating inflammatory mechanisms in both human and

murine macrophage cell lines.

(13,14) This result applied to both

the potentially pathogenic and nonpathogenic species tested.

Low numbers of mycobacteria were detected in the air

of both the nondamaged and moisture-damaged buildings

during the dismantling work. The highest concentration of

mycobacteria was found in the moisture-damaged building,

where a wooden floor was dismantled during the sampling. In

the nondamaged building where mycobacteria were isolated,

the sampling was performed during the dismantling of a

wooden panel wall. The mycobacteria may have been released

from the wooden structures because they have earlier been

isolated from wood-based materials such as wood shavings

and sawdust.(28,29) Mycobacteria may also be found in other

types of wetted building materials. A new, rapid-growing

mycobacterial

species has recently been isolated from the gypsum

board liner of a moisture-damaged day care center.(5,16)

Mycobacteria were found in buildings with both high and

low concentrations of fungi. In fact, mycobacteria were not

found in a moldy building, where the highest concentration

of fungal spores was measured. However, the small number

of samples limits drawing the conclusions of the relationship

between the concentrations of mycobacteria and the fungal and

actinobacterial spore concentrations.

Six-stage impactor sampling proved to be the most successful

method for sampling mycobacteria resulting in mycobacterial

detection in both lowand high concentrations. This

is probably because the bacteria are impacted directly onto

the growth medium without the decontamination step needed

with AGI-30 impinger and filter samples. Decontamination is

an essential part of mycobacterial culture, necessary for the

destruction of more rapid-growing flora.(10) We used 0.5 M

NaOH for the decontamination of the AGI-30 impinger and

filter solutions. However, even this treatment did not completely

inhibit the growth of contaminating microorganisms

found in the air samples of the moisture-damaged buildings.

The lowest yield of mycobacteria was detected with the

AGI-30 impinger. In addition to the losses in the decontamination

phase, some mycobacteria may have escaped

with the bubbling water droplets during the sampling.(30,31)

Furthermore, the detection limit of this method was higher

than in the six-stage impactor sampling. The filter sample in

Site 5 gave the highest concentration of mycobacteria, but the

lowtotal number of samples does not allowfurther conclusions

about the characteristics of the different samplers.

With the six-stage cascade impactor, mycobacteria were

isolated at Stages 1 to 5. The size classes retained by the

Stages from 6 to 1 of the sampler are 0.65–1.1, 1.1–2.1,

2.1–3.3, 3.3–4.7, 4.7–7.0, and >7.0 & #956;m, as given by the

manufacturer (Andersen Samplers, Inc.). This information

indicates that mycobacteria were present in particles small

enough to penetrate the alveolar region of the lungs.

CONCLUSION

Slow-growing mycobacteria are among the microorganisms

that may grow in water-damaged or aged building

materials and be released into the air of worksites during the

dismantling of damaged structures. Because of their potentially

pathogenic and strongly immunostimulating properties, their

health importance as a component of bioaerosol in construction

work deserves further study.

ACKNOWLEDGMENT

This study was supported by a grant from the FinnishWork

Environment Fund.

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