Amplified-fragment length polymorphism fingerprinting of Mycoplasma species

Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprint ing method based on selective amplification of restriction fragments. The p otential of the method for the characterization of mycoplasmas was investig ated in a total of 50 strains of human and animal origin, including Mycopla sma genitalium (n = 11), Mycoplasma pneumoniae (n = 5), Mycoplasma hominis (n = 5), Mycoplasma hyopneunmoniae (n = 9), Myco plasma flocculare (n = 5), Mycoplasma hyosynoviae (n = 10), and Mycoplasma dispar (n = 5), AFLP templ ates were prepared by the digestion of mycoplasmal DNA with BglII and MfeI restriction endonucleases and subsequent ligation of corresponding site-spe cific adapters. The amplification of AFLP templates with a single set of no nselective primers resulted in reproducible fingerprints of approximately 6 0 to 80 fragments in the size range of 50 to 500 bp, The method was able to discriminate the analyzed strains at species and intraspecies levels as we ll, Each of the tested Mycoplasma species developed a banding pattern entir ely different from those obtained from other species under analysis, Subtle intraspecies genomic differences were detected among strains of all of the Mycoplasma species analyzed. The extent of polymorphism varied markedly be tween the analyzed mycoplasmas, comprising pattern similarity levels from 6 1.7% detected among M. dispar strains to 95.9% detected among M. genitalium strains. The results of the present study pro,ide evidence of the high dis criminatory power of AFLP analysis, suggesting the possible applicability o f this method to the molecular characterization of mycoplasmas.