J. Med. Microbiol. -- Vol. 51 (2002), 1117-1127
© 2002 Society for General Microbiology
ISSN 0022-2615
Improved molecular identification of Thermoactinomyces spp. associated with mushroom worker's lung by 16S rDNA sequence typing
JIRU XU,
J.R. RAO*,
B. CHERIE MILLAR,
J. STUART ELBORN
,
,
JAMES EVANS
,
JOHN G. BARR and
JOHN E. MOORE
Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, *Applied Plant Science Division, Department of Agriculture and Rural Development, Newforge Lane, Belfast BT9 5PX, Department of Respiratory Medicine, Belfast City Hospital, Belfast BT9 7AD, Department of Respiratory Medicine, Queen's University of Belfast, School of Medicine, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BA and Regional Mycology Reference Laboratory, Department of Microbiology, The Royal Group of Hospitals, Kelvin Building, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BA
Corresponding author: Dr J. E. Moore (e-mail: jemoore{at}niphl.dnet.co.uk).
Received 20 June 2002; accepted 12 Aug. 2002.
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Abstract
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Mushroom worker's lung (MWL) is a hypersensitivity pneumonitis or allergic alveolitis caused by a type III IgG-mediated immunopathogenic inflammatory reaction in the host due to the inhalation of several thermophilic organisms, including Thermoactinomyces spp. It is difficult to distinguish phenotypically the eight species of this genus; therefore, this study sought to develop an improved molecular means of identifying Thermoactinomyces spp. associated with MWL by partial 16S rDNA PCR amplification and direct sequencing. Hypervariable regions within the 16S rRNA gene, which could be employed as signature sequences of the eight individual species, were identified and employed with highly conserved flanking primers to allow initial PCR amplification, before direct DNA sequencing of the 16S rDNA amplicons. A novel 24-mer 16S rDNA oligonucleotide upstream primer was designed from in silico alignments of all Thermoactinomyces spp. and was employed in combination with downstream (reverse) 16S rDNA primers. This permitted the successful identification of all four isolates associated with mushroom workers’ lung. The method may be useful in the identification of Thermoactinomyces spp. associated with allergic alveolitis or pneumonitis associated with occupational exposure in agricultural and horticultural environments.
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Introduction
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Mushroom worker's lung (MWL) is a hypersensitivity pneumonitis or allergic alveolitis caused by a type III IgG-mediated immunopathogenic inflammatory reaction in the host due to the inhalation of several thermophilic organisms, including members of the genera Thermoactinomyces, Saccharomonospora and Micropolyspora faeni [1]. Of these, Thermoactinomyces spp., including T. vulgaris, have been associated frequently with the disease [2, 3]. The condition, as the name suggests, is prevalent in workers involved in the mushroom industry, particularly in those whose occupation involves working with phase II compost or in the spawning function of mushroom production, because of exposure to high concentrations of aerial mycelia and endospores.
Laboratory culture of causal agents of MWL may be performed from clinical specimens, including sputum and broncho-alveolar lavage (BAL) fluid, as well as from the occupational areas with greatest mycelial and spore loading, by selective culture at elevated temperature (56°C for 4–5 days), although serological detection is most commonly employed. However, reliable identification and, in particular, speciation within the Thermoactinomyces genus, may be troublesome because of the lack of well-defined phenotypic assays to differentiate between the eight species currently described as belonging to this genus [4]. Furthermore, correct identification is important for epidemiological reporting, as well as for identifying strain types that may be employed for subsequent antigen extraction for serological precipitin testing in routine diagnostic laboratories.
Therefore, the aim of this study was to examine partial 16S rDNA PCR amplification and direct sequencing as an improved molecular means of identification of Thermoactinomyces spp. associated with MWL.
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Materials and methods
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PCR primer selection
The complete 16S rDNA sequences of the eight species of Thermoactinomyces [4], including the following organisms: T. candidus (GenBank accession no. AF138732), T. dichotomicus (AF138733), T. intermedius (AF138734), T. peptonophilus (AF138735), T. putidus (AF138736), T. sacchari (AF138737), T. thalpophilus (AF138738) and T. vulgaris (AF138739), were aligned by employing the Clustal method software (DNAstar, Wisconsin, USA) as shown in Fig. 1. Universal 16S rDNA PCR primers were selected to span at least two-thirds of the 16S rDNA gene, to include most regions of hypervariability between the species lying between position 314 and 1370, and included the reverse primers PSR [5] and P13P [6], as described in relation to T. vulgaris (AF138739) (Fig. 2). A novel forward primer, XB1, with the sequence 5'-CAG ACT CCT ACG GGA GGC AGC AGT-3' was designed from the complete 16S rDNA alignment of the eight species and was selected at position 314–337 (T. vulgaris AF138739) which represented 100% sequence homology among all eight species (Fig. 2).
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Fig. 1. Nucleotide alignments of part of the 16S rRNA gene of eight species of Thermoactinomyces. The consensus sequence is shown along the title bar and only differences are displayed.
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Fig. 2. Primer location, amplicon size and nucleotide sequence of the XB1/PSR and XB1/P13P primer combinations used.
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Extraction and PCR amplification of microbial DNA
Four unidentified isolates associated with MWL were examined. All were purified on Colombia agar base (CM331, Oxoid) supplemented with defibrinated horse blood (Oxoid) 5% v/v and D-glucose 1% w/v and were incubated at 56°C for 4–5 days. Genomic DNA was extracted from a single colony with the Roche High Purity PCR Template kit (Roche Diagnostics), in accordance with the manufacturer's instructions. All reaction mixes were set up in a PCR hood in a room separate from that used to extract DNA and the amplification and post-PCR room to minimise contamination, in accordance with the laboratory guidelines of Millar et al. [7]. PCR reaction mixes (50 µl) contained: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 µM (each) dATP, dCTP, dGTP and dTTP, 1.25 U of Taq DNA polymerase (Amplitaq; Perkin Elmer), 0.2 µM (each) of the 16S rRNA primers, as detailed (Fig. 2) and 4 µl of DNA template containing c. 50 ng of DNA/ml of extract. Following a ‘hot start', the reaction mixtures were subjected to the following thermal cycling conditions in a Perkin Elmer 2400 thermocycler: 96°C for 3 min followed by 40 cycles of 96°C for 1 min, 55°C for 1 min, 72°C for 1 min, followed by a final extension at 72°C for 10 min. During each run, molecular grade water (Biowhittaker) instead of DNA was included randomly as a negative control and Staphylococcus aureus DNA was included as a positive control. After amplification, portions (15 µl) were removed, electrophoresed (80 V, 45 min) in agarose (Gibco) gels 2% w/v in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.3) and stained with ethidium bromide (55muµg/1005muml). Gels were visualised under UV illumination with a gel image analysis system (UVP Products) and all images were archived as digital (*.bmp) graphic files.
Sequencing of amplicons and analysis of sequence data
Amplicons for sequencing were purified with the QIAquick PCR purification kit (Qiagen) and eluted in Tris-HCl (10 mM, pH 8.5) before sequencing. Cy-5'-labelled primer, XB1, was prepared and used for sequencing in the forward direction with the ALF Express II (Amersham-Pharmacia, Bucks) employing the Thermo Sequenase fluorescent-labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech; no. RPN 2438) (96°C for 1 min, followed by 25 cycles of 96°C for 10 s, 50°C for 5 s, 60°C for 5 s, followed by a 4°C hold). The sequences obtained were compared with those stored in the GenBank data system with BLAST alignment software (http://www.blast.genome.ad.jp/). Sequence homology identity was determined in accordance with the criteria described previously [8].
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Results and discussion
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All four unknown thermophilic isolates examined generated PCR products of the expected size of c. 762 bp and 1056 bp for primer combinations XB1/PSR and XB1/P13P, respectively (Fig. 3). Subsequent sequence analysis of the 1056-bp amplicon identified the isolates as shown in Table 1 and the sequences have been deposited in GenBank, as detailed (Table 1).
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Fig. 3. PCR amplification of a partial 16S rDNA region (1056 bp) of four unidentified thermophilic organisms associated with mushroom worker's lung. Lanes 1–6, XB1/P13P primer combination; M, 100-bp mol. wt marker (Life Technologies, Paisley, Scotland). Lane 1, RVH210302; 2, RVH210302B; 3, RVH210302C; 4, RVH210302A; 5, negative control; 6, positive PCR control (S. aureus).
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Table 1. Identification of four unidentified thermophilic isolates associated with mushroom worker's lung by PCR amplification and direct sequencing of partial regions of 16S rDNA
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In this study, the initial objective was to identify a highly conserved upstream region of the 16S rRNA gene in Thermoactinomyces spp. that could act as the forward primer in a PCR reaction, in combination with a highly conserved downstream 16S rDNA primer. The approach adopted was to identify hypervariable regions within the 16S rRNA gene, which could be employed as signature sequences of the eight individual species within this genus and to employ highly conserved flanking primers to allow initial PCR amplification, before direct DNA sequencing of the 16S rDNA amplicon.
Several universal 16S rDNA primers have been described previously including PSR [5] and P13P [6]. The sequence regions they detect were also highly conserved within Thermoactinomyces spp. and hence were suitable for PCR detection of this genus. Examination of the 16S rRNA gene sequence between the eight species demonstrated several hypervariable and conserved regions (Fig. 1). In particular, four hypervariable regions, at positions 400–450, 810–840, 1100–1150 and 1220–1250, where base diversity in the 16S rRNA gene was highest, were noted. To exploit this heterogeneity for identification purposes, a conserved primer site was sought upstream of position 400. The primer XB1, a 24-mer oligonucleotide, was identified at position 314–337, with reference to T. vulgaris AF138739, which was totally conserved within the other seven Thermoactinomyces spp. This primer was combined separately with the reverse primers PSR and P13P, as shown in Fig. 3, with the successful generation of PCR amplicons of the expected sizes. Identification of the MWL isolates was subsequently achieved by direct sequencing of the PCR amplicons with the forward primer (XB1) and gave the results shown in Table 1. BLAST alignment was unable to separate T. candidus from T. vulgaris, or T. thalpophilus from T. sacchari. However, recent studies by Yoon et al. [4] based on DNA–DNA hybridisation and 16S rDNA homology have demonstrated that T. candidus is a synonym of T. vulgaris and T. thalpophilus is a synonym of T. sacchari (Fig. 4).
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Fig. 4. Phylogenetic relationship of the eight described Thermoactinomyces spp. based on complete 16S rRNA gene sequences.
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Overall, the use of the XB1/P13P primer combination is recommended, as this will generate a longer PCR amplicon than that generated by the XB1/PSR primer combination and, more importantly, contains a further two hypervariable regions at base positions 1100–1150 and 1220–1250. In conclusion, this study has identified 16S rDNA PCR primer pairs that may be useful in the identification of Thermoactinomyces spp. associated with allergic alveolitis or pneumonitis.
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References
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Abstract
Introduction
