Abstract
BackgroundMycoplasmas 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.MethodsBased 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.ResultsIn silico digestion with the restriction endonuclease AluI (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 AluI, other species gave undistinguishable patterns. For these, an additional restriction digestion, typically with BfaI (C^TAG) or HpyF10VI (GCNNNNN^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.ConclusionTheoretically, 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.
Highlights
Mycoplasmas are present worldwide in a large number of animal hosts
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 amplified rDNA restriction analysis (ARDRA) profiles were only obtained if the sequences of both rrn operons were taken into account
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
Summary
Mycoplasmas are present worldwide in a large number of animal hosts. Already over 100 different species have been identified and characterized and their number increases as more hosts are sampled. Mycoplasmas are phylogenetically related to gram-positive bacteria with low GC-content and belong to the class of the Mollicutes. They form a unique group of bacteria that lack a cell-wall and that contain sterols in their cytoplasmatic membrane. They are of great importance, since several species are pathogenic to animals or humans, whereas species of other mollicute genera infect plants and insects [1]. Already over 100 species have been described, and their number, as well as the number of different hosts is still increasing
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