FEMS Microbiology Ecology 49 (2004) 343–347 www.fems-microbiology.org Environmental mycobacteria from alpine and subalpine habitats Marie-Francoise Thorel a, Joseph O. Falkinham, III b,*, R.G. Moreau c a AFSSA-Alfort, BP 67, 22 rue Pierre Curie, 94703 Maisons-Alfort Cedex, France b Department of Biology, Virginia Polytechnic Institute, State University, Blacksburg, VA 24061-0406, USA c Universite Paris XII, 94000 Creteil, France Received 29 January 2004; received in revised form 5 April 2004; accepted 9 April 2004 First published online 18 May 2004 Abstract Mycobacteria were isolated from a variety of materials such as soil, peat, humus, tufa, sphagnum, and wood, collected in alpine and subalpine habitats. Mycobacteria, including Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium szulgai, Mycobacterium gordonae, Mycobacterium terrae, Mycobacterium chelonae, and Mycobacterium fortuitum were recovered from 69 of 81 (85%) samples. All of the isolates were recovered on medium incubated at 20 and 30 °C. None were recovered if the medium was incubated at 37 °C. The isolation of mycobacteria confirms the presence of these opportunistic pathogens in alpine habitats. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Environmental mycobacteria; Alpine habitats; Growth temperature 1. Introduction tion to environmental extremes. Mycobacteria have been shown to grow over a wide range of pH [12–14], A wide variety of different mycobacterial species have temperature [15,16], and organic matter concentration been recovered from soil, water, vegetables, and dust [1– [15]. Therefore, it would be expected that mycobacteria 4]. In addition to water, soil is a likely reservoir of these could be recovered from extreme environments. Myco- environmental opportunistic mycobacteria [3,5]. Soil bacteria have been recovered from acidic forest soils in has yielded almost every mycobacterial species, with the Finland [5] and from coastal acidic swamps of the exception of members of the Mycobacterium tuberculosis eastern coast of the United States [17]. It is also likely complex [1,3–5]. The mycobacteria that occur most that mycobacteria are present in alpine environments frequently in soil are Mycobacterium fortuitum, Myco- and capable of cold-adaptation. For example, a psy- bacterium gordonae, Mycobacterium nonchromogenicum, chrotrophic strain of Rhodococcus has been reported and Mycobacterium terrae [1,3,6–8]. M. gordonae and [18]. Further, a homologue of the mycobacterial nidA Mycobacterium chelonae have been recovered from gene was amplified by PCR from DNA recovered from sphagnum vegetation [9], soils rich in sphagnum (e.g., pristine and hydrocarbon-contaminated alpine soils [19]. peats) [1,5], and from water draining from peats [10]. In fact, based on the presence of the mycobacterial nidA Mycobacterium malmoense has also been recovered from gene, it was postulated that mycobacteria might be soil [11]. stable members of microbial populations in resource- Recovery of the environmental opportunistic myco- limited habitats [19]. In this work, we report the recov- bacteria from the wide diversity of habitats reported in ery and identification of mycobacteria from a variety of the literature suggests that they are capable of adapta- samples collected in the alpine and subalpine region of Europe. The long-range objective of these investigations * Corresponding author. Tel.: +1-540-231-5931; fax: +1-540-231- is to identify the adaptations leading to survival of 9307. mycobacteria in a nutrient-poor, low temperature E-mail address: jofi[email protected] (J.O. Falkinham, III). region. 0168-6496/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.04.016 344 M.-F. Thorel et al. / FEMS Microbiology Ecology 49 (2004) 343–347 2. Materials and methods all culture, enzymatic, and biochemical tests were per- formed at 20 °C. There were no differences in the reac- 2.1. Environmental samples tions of the culture, enzymatic, or biochemical tests at 20 and 37 °C for the reference strains. However, longer Samples were collected from three Alpine regions, the incubation periods were required (up to 21 days). Jura Forest (peat bogs and meadows), the Vosges For- Comparison of mycolic acid profiles was performed as est, and the Mount White Massif and a subalpine re- described by Goodfellow and Magee [22] and David gion, the Belledone Massif. A total of 81 samples were et al. [23], and carried out on cells that had been grown collected: 44 samples of soil, 8 of peat, 11 of humus, 4 of at 20 °C. For some isolates, identification was confirmed tufa (porous rock), 8 of sphagnum, and 6 of rotting by comparison of patterns of BstEII and HaeII-restric- wood (Table 1). Peat is acidic, partly decomposed plant tion fragments of the PCR amplification product of the remains that accumulate in hollows and wherever the hsp-65 (65-kDa, heat shock protein) gene [24,25]. earth is water-saturated. Humus is organic matter in soils formed from the decomposition of the tissues of dead plants and animals. Samples were collected from 3. Results multiple areas within a site and mixed to produce composite samples. All were collected in the spring 3.1. Recovery of mycobacteria (May) and the ambient air temperature was 15 °C. After collection and preparation of composite samples, all Mycobacteria were recovered from every type of were stored at 4 °C. sample from the different sites and from 69 of the 81 (85%) samples collected. From 81 samples treated with 2.2. Isolation of mycobacteria 0.75% (w/v) HPC without enzymatic treatment, 67 (82.7%) yielded mycobacteria. From the same 81 sam- Sample (2 g) was suspended in 20 ml sterile distilled ples treated with HPC and cellulase, hyaluronidase and water and ground with sterile sand. The particulate amyloglucosidase 69 (85%) yielded mycobacteria. Al- material was allowed to settle on the laboratory bench though addition of enzymes to the isolation regimen for 1 h and the supernatant suspension collected (18 only resulted in a modest increase in the number of ml) to which 20 ml of 0.75% (w/v) hexadecylpyridinium samples yielding mycobacteria (above), the number of chloride (HPC) was added. After incubation at room isolates per sample was significantly (v2, p < 0:05) in- temperature for 18 h, the suspension was centrifuged creased from 74 to 120 isolates from the 81 samples by (5000g for 20 min) and the supernatant liquid discarded. polysaccharide use. This was particularly marked for The pellet was suspended in 1 ml of sterile distilled water soil samples, where polysaccharidase use increased the and distributed on Lowenstein–Jensen (LJ) medium number of isolates from 43 to 74 for the same soil slopes [20]. Because the objective was to isolate myco- samples. bacteria present in alpine and subalpine habitats, the LJ A majority of the isolates (110 of 138, 80%) were slopes were incubated at 20 and 30 °C, in addition to 37 recovered from LJ slopes incubated at 20 °C, whereas °C. To increase the number and variety of mycobacteria, only 20% were recovered at 30 °C (28 of 138). None polysaccharidases were used to release mycobacteria were recovered at 37 °C. The isolates recovered on LJ from particulate and colloidal matter in samples [20,21]. medium incubated at 20 °C failed to grow upon imme- Earlier studies had shown that enzymatic treatment did diate subculture at 37 °C, with the exception of 4 M. not reduce colony counts of cultures or recovery of fortuitum isolates. Mycobacteria were recovered from mycobacteria from soils [20,21]. In fact, the total num- samples whose pH was from 3.1 to 8.3 and whose ber and variety mycobacteria isolated was increased moisture content ranged between 19% and 80%. The [20,21]. Here, it was discovered that hyaluronidase re- highest numbers of isolates were recovered from samples duced the colony forming ability of only one strain of whose pH was below 5 (63%). M. chelonae by 10%. Further, hyaluronidase exposure led to fragmentation of clumps of M. chelonae, but did 3.2. Identification of mycobacteria not disrupt clumps of other mycobacterial species. The identification of 102 of the 120 isolates was de- 2.3. Identification of mycobacteria termined and the isolates were found to belong to eight species of mycobacteria, notwithstanding some differ- Mycobacteria were identified by standard culture, ences observed between the characteristics of the alpine enzymatic, and biochemical tests [22,23], by comparison and subalpine isolates and those of the type strains: 60 to reference strains. Because the majority of isolates M. chelonae,14M. malmoense,11M. gordonae,7M. were recovered at 20 °C and none were isolated at 37 °C, fortuitum,5Mycobacterium szulgai,4Mycobacterium M.-F. Thorel et al. / FEMS Microbiology Ecology 49 (2004) 343–347 345 Table 1 Identification of mycobacteria by sample type and origin Species Soil Humus Tufa Peat Rotting wood Sphagnum Total M. chelonae 42a;b 5a;c 1a 2c 5a;b 5c;d 60 M. fortuitum 5a;d 1a 000 1a 7 M. gordonae 2a 1a 3a 3a;c 02a 11 M. malmoense 12a;d 1a;c 000 1a 14 M. szulgaı03a 01a 01a 5 M. kansasii 0001c 001 M. kansasii, non-pigmented 1d 00 1a 01d 3 M. terrae complex 0 1a 000 0 1 Unidentified mycobacteria 12a;d 1c 001a 4a;d 18 Total 74 13 4 8 6 15 120 a Jura Forest. b Vosges Forest. c Subalpine Forest. d Alpine moor. kansasii (3 unpigmented), and 1 M. terrae complex 4. Discussion (Table 1). Species assignment of 18 of the 120 isolates (15%) could not be made in keeping with other studies Mycobacteria were isolated from every type of sam- of environmental mycobacteria [5–7,10]. The M. chelo- ple in each of the different alpine and subalpine habitats, nae isolates were all non-pigmented, did not reduce ni- demonstrating that the environmental opportunistic trate, were arylsulfatase- and catalase-positive, and mycobacteria are normal alpine and subalpine inhabit- produced mycolic acids I and II [22,23].