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Applied and Environmental Microbiology, December 2006, p. 7602-7606, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.00930-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Relationships between Mycobacterium Isolates from Patients with Pulmonary Mycobacterial Infection and Potting Soils{triangledown} ,{dagger}

Mary Ann De Groote,1 Norman R. Pace,1* Kayte Fulton,2 and Joseph O. Falkinham III3

Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347,1 National Jewish Medical and Research Center, Denver, Colorado 80206,2 Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-04063

Received 19 April 2006/ Accepted 5 October 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
High numbers of mycobacteria, including known pathogenic species such as Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chelonae, were recovered from aerosols produced by pouring commercial potting soil products and potting soil samples provided by patients with pulmonary mycobacterial infections. The dominant mycobacteria in the soil samples corresponded to the dominant species implicated clinically. Profiles of large restriction fragments obtained by pulsed-field gel electrophoresis demonstrated a closely related pair of M. avium isolates recovered from a patient and from that patient's own potting soil. Thus, potting soils are potential sources of infection by environmental mycobacteria. Use of dust-excluding masks should be considered during potting or other activities that generate aerosol with soil.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mycobacterium avium and a variety of other Mycobacterium species are opportunistic human pathogens that occur in the environment (2, 3). On the basis of the identity of pulsed-field gel electrophoresis (PFGE) profiles of large genome restriction fragments, it has been shown that M. avium isolates recovered from AIDS patients were clonally related to isolates recovered from water to which the patients had been exposed (15). Because opportunistic mycobacteria are ubiquitous in the environment (3), it is likely that other sources of infection exist, such as soil. Peat-rich boreal forest soils of Finland, for instance, contain high numbers of mycobacteria (4), and this has been associated with the high frequency of mycobacterial infection in AIDS patients (10) and with the annual number of Finnish patients diagnosed with M. avium, M. intracellulare, and Mycobacterium malmoense infections (5). Another potential source of mycobacteria is potting soils. Soil samples collected from potted plants were shown to yield mycobacteria frequently (89%), including Mycobacterium avium (27%) and Mycobacterium intracellulare (28%). Moreover, freshly opened bags of commercial potting soil also contain M. avium and M. intracellulare (17). Commercial potting soils contain a substantial proportion of sphagnum vegetation (i.e., peat), which can contain high numbers of mycobacteria (11) and supports the growth of mycobacteria (6).

Over the past decades in the United States, the frequency of slender, elderly women and men with pulmonary mycobacterial infections caused by Mycobacterium avium, Mycobacterium intracellulare, and other mycobacteria has increased (7-9). These patients typically lack predisposing factors such as chronic obstructive pulmonary disease, bronchiectasis, or pneumoconiosis that are commonly associated with pulmonary mycobacterial infections (7, 8). A number of such patients had been seen by referral at the National Jewish Medical and Research Center, and a significant proportion were gardeners. In order to determine the quantities and identities of soil mycobacteria in aerosols to which patients were exposed, we conducted culture and molecular analyses of mycobacteria from soil samples provided by cooperating patients in order to determine if soils are potential sources of nontuberculous mycobacterial infections.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Commercial potting soils.
Two commercial potting soils were purchased and used in this study; we designated these commercial soil 1 and commercial soil 2. Both contained sphagnum peat moss along with other ingredients. A freshly opened bag of commercial soil 2 was used, whereas the commercial soil 1 package had been opened for approximately 1 month and the material was considerably drier.

Patients and soil samples.
Patients were recruited from the Mycobacterial Clinical Service at the National Jewish Medical and Research Center. This center is a large referral hospital and clinic in Denver, Colorado, which sees approximately 250 patients per year (predominantly from the United States) with a variety of nontuberculous mycobacterial infections. From the experience of one of the authors (M.A.D.), it was noted that a sizeable proportion of the patients seen were women (~80%) and also gardeners. Sequential patients seen at the National Jewish Medical and Research Center over a 1- to 2-year period were enrolled if they (i) had a history of gardening and (ii) were willing to participate in the study. Approval for the study was granted by the Institutional Review Board (protocol HS 1427). Over a period of 1 year, sequential patients referred to the National Jewish Medical and Research Center that had soil exposure were asked to give informed consent to provide soil for the study. The choice of soils was left to the individual patients, who provided 1 to 2 cups of soil to which they had been exposed recently as a result of plant potting or other gardening activities. The soils were mailed to the National Jewish Medical and Research Center after the patients returned to their homes. All patient samples were numbered to prevent disclosure of personal information.

For a list of the potting soil samples collected by the individual patients, see Table S1 in the supplemental material. Twenty-one of the 26 patients in the study group submitted 81 personal potting soil samples, of which 79 were analyzed. Those not analyzed were predominantly rock and gravel.

Patient mycobacterial isolates.
Mycobacterial isolates from respiratory specimens were obtained in the course of routine patient care. Identification of each isolate was confirmed by the National Jewish Medical and Research Center Clinical Mycobacteria Laboratory according to standard procedures (e.g., morphology, growth rate, high-performance liquid chromatography patterns, and/or identification with a commercial DNA probe [GenProbe; AccuProbe, San Diego, CA]). Clinical isolates were identified in parallel with the soil isolates as described in the section on the identification and enumeration of mycobacteria below. This PCR restriction enzyme pattern analysis method of identification was done as described by Telenti et al. (13). Briefly, amplification of a 441-bp fragment of the 65-kDa heat shock protein gene (hsp65) was followed by restriction endonuclease digestion with HaeIII and BstEII. Strain identity was done by visually comparing the pattern of HaeIII and BstEII restriction fragments to patterns from known species of mycobacteria.

Aerosol collection medium.
The agar medium employed for collection of aerosolized particulates was Middlebrook 7H10 agar medium (M7H10; Difco Becton Dickinson, Sparks, MD) containing 0.5% (vol/vol) glycerol (G) and 10% (vol/vol) oleic albumin (OA) enrichment. To retard the growth of other bacteria and fungi, malachite green was added to a final concentration of 0.001% (wt/vol) to the agar medium (M7H10-MG). Cultures were incubated for 17 days at 37°C and inspected weekly for the appearance of mycobacterium-like colonies.

Collection of aerosols.
Pulmonary infection results when potential pathogens are associated with particles sufficiently small to penetrate the deeper airways. Consequently, we tested the aerosolization of soil mycobacteria under conditions designed to mimic the activities of a gardener. A ring funnel support and a funnel (14 cm at the top with a 3-cm-diameter hole) on a ring stand were placed in an enclosed glove box, and the height was adjusted such that the distance from the bottom of the funnel to the surface of a collection pan was 30 cm. A stopper was placed in the hole at the bottom of the funnel, and 100 g of commercial potting soil or 2 to 100 g of each patient's potting soil (depending upon the amount of the sample obtained) was transferred into the funnel without generating dust. Six petri dishes containing 25 ml of M7H10 or M7H10-MG agar were inserted into a stoppered six-stage Andersen cascade sampler (1), and the loaded six-stage Andersen Cascade sampler was transferred into the glove box. The distance from the funnel and collection pan to the Andersen sampler was approximately 30 cm. The pump for the sampler remained outside the hood. The stopper was removed from the Andersen cascade sampler, the pump was turned on (28.3 liters/min), and the stopper was removed from the bottom of the funnel to allow the potting soil to pour through the funnel into the collection pan (thus generating a particulate aerosol). Aerosols were collected for 10 min, and the plates were recovered from the Andersen sampler. Plates were sealed with Parafilm and incubated at 37°C for 17 days. Between aerosol experiments, the glove box was decontaminated by washing with 4% (vol/vol) Lysol and allowing the disinfectant solution to remain on the walls for at least 15 min. After washing, a germicidal UV light in the glove box was illuminated overnight. The funnel and all supports were sterilized by autoclaving between aerosolization experiments. Three separate control experiments demonstrated that aerosol samples collected after pouring sterilized potting soil following that decontamination regimen did not yield any mycobacteria or other microorganisms.

Identification and enumeration of mycobacteria.
Every putative mycobacterial colony appearing on M7H10 agar or M7H10-MG agar was streaked for isolation on M7H10 agar. Acid-fast colonies of the same morphology and appearance were counted, and a representative colony was transferred to M7H10 agar slants for archiving and identification. DNA was isolated from acid-fast isolates by suspending 1 mg of cell mass in a tube with 1 ml of water and 0.1 g of 0.1-mm-diameter glass beads and shaking the mixture in a Bead-Beater (Bio-Spec Products, Bartlesville, OK) for 1 min at the maximum setting. The glass beads and cellular debris were pelleted by centrifugation (5,000 x g for 5 min), and the DNA-containing supernatant was collected. Identification of isolates as members of the genus Mycobacterium and as M. avium or M. intracellulare was performed by a nested PCR (16). PCR amplification of the hsp-65 gene and the pattern of HaeIII and BstEII restriction fragments as described by Telenti et al. (13) identified other species and confirmed the identification of M. avium and M. intracellulare isolates. Ribosomal operon sequencing (including the partial 16S rRNA gene and the 3' end of the 23S rRNA gene) was performed on the eight isolates subjected to PFGE. The ribosomal gene primers used were small-subunit primer 8F 5'-AGAGTTTGATCCTGGCTCAG-3' and large-subunit primer 2654R 5'-CCGGTCCTCTCGTACT-3'. A Sigma AccuTaq kit was used. The PCR conditions used were 95°C for 3 min; 30 cycles of 94°C for 1 min, 50°C for 2 min, and 68°C for 4 min; and 68°C for 20 min after addition of 1 µl of deoxynucleoside triphosphates (2.5 mM each) and 1 U of our laboratory's Taq polymerase. PCR products were separated on a 0.8% agarose gel, and bands were cut out and gel purified by Millipore Montage gel extraction according to the manufacturer's instructions. They were then cloned into the TOPO vector (Invitrogen) and sequenced. DNA sequencing of the amplicons was performed with T3 and T7 standard sequencing primers by dideoxy chain termination chemistry according to the manufacturer's (GE Healthcare, Piscataway, NJ) specifications, and reactions were run on a MegaBACE 1000 automated sequencing instrument according to the manufacturer's recommendations. Sequence comparisons were made by searching BLAST.

PFGE.
Following inoculation from single colonies, mycobacterial isolates were inoculated into 2 ml of M7H9 broth (Difco Becton Dickinson, Sparks, MD) containing 0.5% (vol/vol) G and 10% (vol/vol) OA in a screw-cap tube (16 by 125 mm) and incubated without shaking at 37°C for 4 days or until turbid. Mycobacteria grow better without shaking because of their sensitivity to oxygen at a low inoculum density (J.O.F., personal experience). One milliliter of the inoculum was used to inoculate 9 ml of M7H9-G-OA contained in a 250-ml sidearm flask. After 5 days of incubation at 37°C (late log phase), filter-sterilized solutions of D-cycloserine (10 mg/ml) and ampicillin (1 mg/ml) were added to final concentrations of 1 mg/ml and 0.1 mg/ml (respectively). These two antibiotics weaken the cell wall and result in higher yields of DNA. Incubation was continued until the turbidity reached an absorbance of 2.1 to 2.3 (540 nm). Cells were collected by centrifugation (5,000 x g for 20 min) of the 10-ml culture volume, suspended by vortexing in 10 ml of water, and pelleted again to improve DNA yields. After that washing, the cells were suspended in 5 ml of sterile distilled water. A portion of the washed cells (250 µl) was mixed with 250 µl of dissolved and cooled 1% (wt/vol) InCert agarose (Cambrex BioScience, Rockland, ME) and transferred into disposable plug molds. Cells in the agarose plugs were exposed to 2 mg lysozyme/ml in Tris-EDTA buffer at 37°C overnight, followed by exposure to 0.5 mg of proteinase K/ml and 1% (wt/vol) sodium dodecyl sulfate at 55°C for 48 h. Proteinase K was inactivated by two washes with 1 mM phenylmethylsulfonyl fluoride, followed by two washes in Tris-EDTA buffer. All chemicals were obtained from Sigma-Aldrich, Inc. (St. Louis, MO). DNA in agarose plugs was digested with XbaI, and fragments were separated in 1% (wt/vol) SeaKem Gold agarose (Bio-Rad Laboratories, Inc., Hercules, CA) in 0.5x Tris-borate-EDTA buffer (pH 8.0) (10a) in a CHEF-DR II system (Bio-Rad Laboratories, Hercules, CA) in 0.5x Tris-borate-EDTA buffer (pH 8.0) at 6 V/cm at 14°C with switch times of 5 to 30 s ramped linearly over 15 h. A 120° angle was used. DNA fragments were photographed over a UV light after staining with 0.5 µg of ethidium bromide/ml for 1 h, followed by destaining in tap water for 1 h.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Aerosolization of mycobacteria from commercial potting soils.
As detailed in Materials and Methods, samples of two commercial potting soils were dropped into a collection pan and aerosols were collected (283 liters) onto agar growth medium selective for mycobacteria in a six-stage Andersen cascade sampler located 30 cm from the soil. Aerosol collection was limited to 10 min to mimic the actions and exposure of someone potting plants. Results of colony counts are summarized in Table 1. Similar numbers of morphologically identical colony-forming isolates were recovered from commercial potting soil 2 in stages 1, 2, and 3 in the duplicated aerosol collection experiments. More acid-fast isolates were recovered from freshly opened commercial potting soil 2 (two experiments) than from commercial soil 1, possibly because the commercial soil container had been open for 1 month and the potting soil was considerably drier than was commercial potting soil 2. These results show clearly that acid-fast positive bacteria, presumptive mycobacteria, are aerosolized. Acid-fast colonies were recovered from plates in the third (5 µm) through the fifth (2 µm) stages of the Andersen sampler, indicating their association with particles sufficiently small to enter the human lung (1). Because the aim of these experiments was to establish the aerosolization of soil mycobacteria in soil, isolates were not identified further.


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  		TABLE 1. Recovery of acid-fast bacterial colonies from commercial potting soil aerosols

 
Recovery of mycobacteria from patient potting soils.
Mycobacteria from respiratory secretions of the 26 patients enrolled in this study were identified to the species level during clinical diagnosis and are summarized in Fig. 1A. Potting soils were solicited from the patients, 21 of whom provided 82 soil samples, 79 of which were analyzed (Materials and Methods) for aerosolized mycobacteria. Each soil sample (2 to 100 g; mean, 42 g) was subjected to aerosolization, and mycobacterial CFU were captured with the Andersen cascade sampler. Colonies with representative morphologies were identified by DNA analysis (Materials and Methods). The results for all of the soil samples are summarized in Fig. 1B (see also Table S1 in the supplemental material). Particularly conspicuous in Fig. 1A and B is the species correspondence between clinical pathogens and mycobacteria detected in soil aerosols. The most frequent pathogens, such as M. avium and M. intracellulare, were the most abundant among the soil mycobacteria. Most (14/21) of the patients were infected by species detected in their soil samples. Identification to the species level does not establish a direct connection between the patient's disease and a specific soil. The results do, however, show that opportunistic pathogens were abundant in the aerosols from patients' soil samples. Samples contained significant levels of potential pathogens, typically 0.04 to 0.4 CFU/liter of air collected in the Anderson sampler and as high as 2,400/283 liters of air with one sample (see Table S1 in the supplemental material). Mycobacteria were isolated from every stage of the Andersen sampler, particularly stages 4 (71% of the samples), 5 (57% of the samples), and 6 (29% of the samples). In one case, aerosol from potting soil A004-4 yielded 1.87 CFU/liter from stage 4, 0.24 CFU/liter from stage 5, and 0.003 CFU/liter from stage 6, all of which were M. intracellulare and had the same colony morphology.


Figure 1
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  		FIG. 1. (A) Distribution of mycobacterial species from respiratory samples of patients. Isolates from patient sputum or bronchoalveolar lavage fluid. MAC (NOD) is M. avium complex not further identified to the species level. (B) Distribution of mycobacterial species from total soil samples (n = 79) provided by patients.

 
At least one of the potting soil samples obtained from all the patients submitting soil samples yielded mycobacteria, and most samples yielded different species (see Table S1 in the supplemental material). The frequency with which patients were exposed to the different mycobacteria can be estimated by the frequency of occurrence of particular species in at least one of the soil specimens provided by the particular patients. These results are summarized in Fig. 1A and B and 2. It is noteworthy that the most frequently encountered mycobacterial species in the aerosols of potting soils, M. intracellulare and M. avium, are also the main pathogens in nontuberculous mycobacterial pulmonary disease (8).


Figure 2
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  		FIG. 2. Percentages of patients (n = 21) with soil samples that harbor particular species of mycobacteria.

 
Comparison of PFGE profiles of patient and potting soil isolates.
Some of the mycobacteria isolated from patients and their own soil aerosols seemed identical in species identification tests and colony morphology. In order to obtain a more highly resolved identification of some of these patient and soil pairs, we compared the profiles of genomic restriction endonuclease digestion fragments separated by PFGE. Patient and potting soil aerosol isolates compared by PFGE included two instances each of M. avium and M. intracellulare. Each pair was identical over the 16S and 23S rRNA gene fragments sequenced (not shown), confirming the culture and hsp-65 restriction patterns. All of these potting soil aerosol isolates were recovered from either stage 4 or 5 of the Andersen sampler (see Table S1 in the supplemental material), and thus the cells were associated with particles able to enter the human lung.

The results shown in Fig. 3 show that 15 to 25 fragments were produced by XbaI restriction endonuclease digestion, a sufficient number for reliable comparisons to be made (14). One of the pairs of M. avium patient and potting soil aerosol isolates differed by only two bands and thus are closely related on the basis of recommended criteria for strain typing by PFGE (14). The two patient and potting soil M. intracellulare isolates appear to be closely related subtypes, differing by seven and five bands, respectively. The other set of M. avium isolates appeared to be unrelated in this assay.


Figure 3
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  		FIG. 3. PFGE profiles of restriction endonuclease digestion fragments of total DNA from the following isolates (for details of the patient and epidemiologically matched potting soil aerosol isolates, see Table S1 in the supplemental material): M. avium (Mav1) strains A002 (lane 1, patient isolate [P]) and A002-4-4-1 (lane 2, soil isolate [S]), M. intracellulare (Min1) isolates A014 (lane 3, patient isolate) and A014-2-5-2 (lane 4, soil isolate), M. avium (Mav2) isolates A019 (lane 5, patient isolate) and A019-3-4-1 (lane 6, soil isolate), and M. intracellulare (Min2) isolates A020 (lane 7, patient isolate) and A0206-4-2 (lane 8, soil isolate).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recovery of mycobacteria from commercial potting soil aerosols.
The results presented here show clearly that dust generated from commercial potting soil contains a variety of culturable mycobacteria in relatively high numbers in association with aerosols sufficiently small in size to penetrate deeply into pulmonary airways (Table 1). Although here mycobacteria were recovered from aerosols, the results are consistent with those of Yajko et al. showing that samples from freshly opened bags of commercial potting soil yielded culturable mycobacteria (17). Legionella spp. also have been recovered from potting soils (12). The method that we used to generate soil aerosols, pouring through a funnel, is similar to operations routinely performed by gardeners as they pour potting soil into pots. Thus, commercial soils are a source of opportunistic pathogens. The technique appeared reproducible, as evidenced by the similarity in the numbers of acid-fast bacilli and their distribution in the different stages of the Andersen sampler (Table 1). We acknowledge that the method is subject to variation due to many factors, for instance, the character of the soil tested and its moisture content, as well as other factors. Nonetheless, in order to focus on aerosolized mycobacteria, we used this experimental approach to analyze potting soil samples provided by individual patients with pulmonary mycobacterial infections.

Recovery of mycobacteria from patient potting soil aerosols.
As with the commercial potting soil samples, the potting soil samples obtained from patients yielded a variety of mycobacteria (see Table S1 in the supplemental material). We have no information on whether or not the soil samples were from commercial sources. Samples typically yielded 10 to 100 CFU collected, and some samples yielded very high numbers; for instance, in one sample ~2,400 CFU were collected in the 10-min sampling period. Further, with all of the patient soils mycobacteria were recovered from stages of the Andersen cascade sampler that collect particles able to enter the human lung and even the alveoli (i.e., 1 to 5 µm in diameter) (1). For two-thirds (14 of 21) of the patients, the isolates from the patients and from their aerosolized potting soil samples yielded the same species (see Table S1 in the supplemental material). Because only a single isolate of each species was obtained from the patients and infection with multiple Mycobacterium species occurs, it is possible that the percentage of species matches is higher.

The characteristics of the different patient potting soil samples and amounts of soil available for analysis were quite varied. Some were relatively moist, some were dry, some contained only peat, and some contained substantial amounts of soil. It is likely that potting soil composition and moisture content directly influence the yield of mycobacteria in aerosols. It was observed that the drier samples generated more aerosols and dust than the moist samples. It is possible that drying, while increasing the propensity to form aerosols and dust, might reduce the number of viable mycobacteria.

Comparison of PFGE profiles of selected pairs of patient and potting soil isolates.
The most frequently encountered mycobacteria in these soil aerosols, M. avium and M. intracellulare, are the most common pathogens recovered from the patients (Fig. 1A and B). Mycobacterial species are highly heterogeneous, however, so we used a genomic fingerprinting method, PFGE restriction analysis, to compare pairs of selected patient and soil isolates. PFGE profiles were compared between four pairs of patient and their aerosolized potting soil isolates of the same species. The potting soil aerosol isolates were recovered from either stage 4 or 5 of the Andersen sampler, and those stages recover mycobacterial cells associated with particles able to enter the human lung and lead to infection.

The PFGE profiles (Fig. 3) showed that one M. avium patient isolate and the corresponding potting soil aerosol isolate differed by only two bands (Fig. 3) and thus appear to be members of the same clone on the basis of the recommended criteria of Tenover et al. (14). The ability to discriminate between pairs is high, based on the production of ~25 fragments by restriction endonuclease digestion of total DNA from the isolates. The M. intracellulare patient and potting soil aerosol isolates from a different patient are related subtypes differing by only seven bands. This comparison of restriction fragments by PFGE was limited by the fact that only a single patient isolate was available for comparison. As a consequence, the clonal diversity of the mycobacterial species infecting the patient could not be determined. If additional patient and potting soil isolates were compared, more or closer matches might be identified. Another limitation of the study was that the patient isolates were recovered from the patients ~1 year before the potting soil samples were obtained and analyzed. Because potting soil supports the growth of mycobacteria (6), it is possible that subclones had emerged in potting soils used by the patient at the time of exposure if sufficient moisture was retained.

Potting soil aerosols as a source of mycobacterial infection.
The data provided in this report do not establish that potting soils were the source of the patient infections. The results, however, do implicate potting soil aerosols as a possible source of mycobacterial infection in the slender, elderly population. Even in those cases where clonality of patient and potting soil aerosol isolates was not shown, it is clear that aerosols and dust generated by dropping a variety of potting soils contained substantial numbers of mycobacteria known for opportunistic pathogenicity. In the absence of knowledge of the minimum infectious dose of aerosolized mycobacteria, it is difficult to extrapolate the results in this report to calculate a probability of pulmonary disease. Such an extrapolation is made more problematic by unknowable factors such as the amounts of potting soil manipulated and the aerosol amounts generated. Nonetheless, the results show that there are ample potentially pathogenic mycobacteria in soils. We acknowledge that this culture-based study likely underestimated the actual mycobacterial concentrations in the soil samples tested, since only a small fraction of environmental microbes are cultured. Even on the basis of the culture results, however, it appears prudent to advise susceptible individuals to wear a dust mask while engaging in gardening activities where potting soils are being used and aerosols are generated.


   ACKNOWLEDGMENTS
 
Applied Microbiology and Genetics provided funds for the aerosolization and identification of mycobacteria at Virginia Tech. We acknowledge Gwen Huitt, Michael Iseman, and the Alfred P. Sloan Foundation for support.

We acknowledge the technical assistance of Myra Williams in performing the aerosolization experiments and identification of mycobacteria and Tracey Sanchez and Leonid Heifets for identification of mycobacteria. We are grateful for the assistance of Allison St. Amand in manuscript preparation.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309. Phone: (303) 735-1808. Fax: (303) 492-7744. E-mail: nrpace{at}colorado.edu. Back

{triangledown} Published ahead of print on 20 October 2006. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Andersen, A. A. 1958. New sampler for the collection, sizing, and enumeration of viable airborne particles. J. Bacteriol. 76:471-484.[Free Full Text]
  2. Arbeit, R. D., A. Slutsky, T. W. Barber, J. N. Maslow, S. Niemczyk, J. O. Falkinham III, G. T. O'Connor, and C. F. von Reyn. 1993. Genetic diversity among strains of Mycobacterium avium causing monoclonal and polyclonal bacteremia in patients with AIDS. J. Infect. Dis. 167:1384-1390.[Medline]
  3. Falkinham, I. J. O. 1996. Epidemiology of infection by nontuberculous mycobacteria. Clin. Microbiol. Rev. 9:177-215.[Medline]
  4. Iivanainen, E. K., P. J. Martikainen, L. Räisänen, and M. L. Katila. 1997. Mycobacteria in boreal coniferous forest soils. FEMS Microbiol. Ecol. 23:325-332.[CrossRef]
  5. Katila, M.-L., E. Iivanainen, P. Torkko, J. Kauppinen, P. Màrtikainen, and P. Väänänen. 1995. Isolation of potentially pathogenic mycobacteria in the Finnish environment. Scand. J. Infect. Dis. Suppl. 98:9-11.[Medline]
  6. Kazda, J. 2000. The ecology of mycobacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands.
  7. Kennedy, T. P., and D. J. Weber. 1994. Nontuberculous mycobacteria. An underappreciated cause of geriatric lung disease. Am. J. Respir. Crit. Care Med. 149:1654-1658.[Abstract]
  8. Prince, D. S., D. D. Peterson, R. M. Steiner, J. E. Gottlieb, R. Scott, H. L. Israel, W. G. Figueroa, and J. E. Fish. 1989. Infection with Mycobacterium avium complex in patients without predisposing conditions. N. Engl. J. Med. 321:863-868.[Abstract]
  9. Reich, J. M., and R. E. Johnson. 1991. Mycobacterium avium complex pulmonary disease. Incidence, presentation, and response to therapy in a community setting. Am. Rev. Respir. Dis. 143:1381-1385.[Medline]
  10. Ristola, M. A., C. F. von Reyn, R. D. Arbeit, H. Soini, J. Lumio, A. Ranki, S. Bühler, R. Waddell, A. N. A. Tosteson, I. J. O. Falkinham, and C. H. Sox. 1999. High rates of disseminated infection due to non-tuberculous mycobacteria among AIDS patients in Finland. J. Infect. 39:61-67.[CrossRef][Medline]
  11. Sambrook, J. E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  12. Schröder, K.-H., J. Kazda, K. Müller, and H. J. Müller. 1992. Isolation of Mycobacterium simiae from the environment. Zentbl. Bakteriol. 277:561-564.
  13. Steele, T. W., J. Lanser, and N. Sangster. 1990. Isolation of Legionella longbeachae serogroup 1 from potting mixes. Appl. Environ. Microbiol. 56:49-53.[Abstract/Free Full Text]
  14. Telenti, A., F. Marchesi, M. Balz, F. Bally, E. C. Böttger, and T. Bodmer. 1993. Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J. Clin. Microbiol. 31:175-178.[Abstract/Free Full Text]
  15. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.[Medline]
  16. von Reyn, C. F., J. N. Maslow, T. W. Barber, I. J. O. Falkinham, and R. D. Arbeit. 1994. Persistent colonisation of potable water as a source of Mycobacterium avium infection in AIDS. Lancet 343:1137-1411.[CrossRef][Medline]
  17. Wilton, S., and D. Cousins. 1992. Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. PCR Methods Appl. 1:269-273.[Medline]
  18. Yajko, D. M., D. P. Chin, P. C. Gonzalez, P. S. Nassos, P. C. Hopewell, A. L. Rheingold, J. C. R. Horsburgh, M. A. Yakrus, S. M. Ostroff, and W. K. Hadley. 1995. Mycobacterium avium complex in water, food, and soil samples collected from the environment of HIV-infected individuals. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 9:176-182.[Medline]

Applied and Environmental Microbiology, December 2006, p. 7602-7606, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.00930-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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