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Improved molecular identification of Thermoactinomyces spp. associated with mushroom worker's lung by 16S rDNA
Jiru Xu, J R Rao1, B Cherie Millar
1Northern 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.
Abstract:
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.
Insights
Mushroom worker's lung (MWL) is an allergic lung condition caused by Thermoactinomyces bacteria. This study developed a new molecular method using DNA sequencing to accurately identify these bacteria, aiding in diagnosis and understanding of occupational lung diseases.
Area of Science:
- Microbiology
- Immunology
- Pulmonary Medicine
Background:
- Mushroom worker's lung (MWL) is a hypersensitivity pneumonitis caused by thermophilic actinomycetes, primarily Thermoactinomyces species.
- Phenotypic identification of the eight Thermoactinomyces species is challenging, hindering accurate diagnosis and etiological understanding.
- Understanding the specific Thermoactinomyces species involved is crucial for managing occupational lung diseases.
Purpose of the Study:
- To develop an improved molecular method for identifying Thermoactinomyces species associated with MWL.
- To utilize partial 16S ribosomal DNA (rDNA) PCR amplification and direct sequencing for species-specific identification.
- To design novel primers for enhanced accuracy in Thermoactinomyces identification.
Main Methods:
- Partial 16S rDNA gene amplification using PCR with specifically designed primers.
- Direct DNA sequencing of the amplified 16S rDNA fragments.
- In silico analysis to identify hypervariable regions and design novel oligonucleotide primers.
Main Results:
- Identification of hypervariable regions within the 16S rRNA gene suitable for species-specific signatures.
- Successful amplification and sequencing of 16S rDNA from Thermoactinomyces isolates.
- Development and validation of a novel upstream primer for improved identification of Thermoactinomyces species.
Conclusions:
- The developed molecular method enables accurate identification of Thermoactinomyces species linked to MWL.
- This technique offers a valuable tool for diagnosing hypersensitivity pneumonitis in occupational settings.
- The findings contribute to understanding and managing allergic alveolitis caused by environmental exposures.