Random amplified ribosomal DNA restriction analysis for rapid identification of thermophilic actinomycete-like bacteria involved in hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is a pulmonary disease characterised by i nflammation that can be caused by, amongst other substances, a subset of 4 thermophilic mycelial bacteria: Saccharopolyspora rectivirgula, Saccharomon ospora viridis, Thermoactinomyces sacchari, and Thermoactinomyces vulgaris. Air sampling analyses in highly contaminated environments are often perfor med to evaluate exposure to these species which are difficult and fastidiou s to identify by conventional techniques. The aim of this study was to use amplified ribosomal DNA restriction analysis (ARDRA) to develop a method of identification for those thermophilic organisms that would be more rapid a nd simple. Strains of these 4 species were obtained from the American type culture collection (ATCC) and were characterized using biochemical tests an d ARDRA patterns obtained on their partial-lenght amplified 16S rDNAs. To v alidate this approach, ARDRA with two restriction enzymes, TaqI and HhaI, w as applied to 49 thermophilic actinomycete-like strains from environmental samples (sawmills). The results obtained show that combining some cultural characteristics and biochemical tests, such as xanthine or hypoxanthine dec omposition, growth in the presence of NaCl, lysozyme or novobiocin, and spo re resistance over 100 degreesC provide a rough identification and selectio n of the genera of interest. Consequently, target species could be confirme d by digestion of partial-lenght 16S rDNA with the use of TaqI and HhaI res triction enzymes that gave specific restriction patterns. ARDRA analyses on the 49 environmental actinomycete-like organisms revealed the presence of 8 Saccharopolyspora rectivirgula, 2 Saccharomonospora viridis, and 15 Therm oactinomyces vulgaris strains, the other strains had restriction patterns d ifferent than those of the species of interest. Results of the present stud y will be applicable to other potential HP environments such as dairy barns , peat bogs and compost plants.