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Background  Mycoplasmas are present worldwide in a large number of animal hosts. Due to their small genome and parasitic lifestyle,  Mycoplasma  spp. require complex isolation media. Nevertheless, already over 100 different species have been identified and characterized and their number increases as more hosts are sampled. We studied the applicability of amplified rDNA restriction analysis (ARDRA) for the identification of all 116 acknowledged  Mycoplasma  species and subspecies.    Methods  Based upon available 16S rDNA sequences, we calculated and compared theoretical ARDRA profiles. To check the validity of these theoretically calculated profiles, we performed ARDRA on 60 strains of 27 different species and subspecies of the genus  Mycoplasma .    Results   In silico  digestion with the restriction endonuclease  Alu I (AG^CT) was found to be most discriminative and generated from 3 to 13 fragments depending on the  Mycoplasma  species. Although 73  Mycoplasma  species could be differentiated using  Alu I, other species gave undistinguishable patterns. For these, an additional restriction digestion, typically with  Bfa I (C^TAG) or  Hpy F10VI (GC^NNGC), was needed for a final identification. All  in vitro  obtained restriction profiles were in accordance with the calculated fragments based on only one 16S rDNA sequence, except for two isolates of  M. columbinum  and two isolates of the  M. mycoides  cluster, for which correct ARDRA profiles were only obtained if the sequences of both  rrn  operons were taken into account.    Conclusion  Theoretically, restriction digestion of the amplified rDNA was found to enable differentiation of all described  Mycoplasma  species and this could be confirmed by application of ARDRA on a total of 27 species and subspecies.

Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of Mycoplasma species