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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.

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.