Evaluation of amplified fragment length polymorphism analysis for inter- and intraspecific differentiation of Mycobacterium bovis, M-tuberculosis, and M-ulcerans

The usefulness of amplified fragment length polymorphism (AFLP) analysis wa s evaluated for the discrimination of Mycobacterium bovis (17 strains), M. tuberculosis (15 strains), and M. ulcerans (12 strains) at the inter- and i ntraspecific level. The AFLP technique is a whole-genome coverage genotypic fingerprinting method based on the selective PCR amplification of modified restriction fragments obtained through a double enzymatic digest and subse quent ligation of double-stranded restriction site-specific adapter oligonu cleotides. Selective amplification of ApaI/TaqI templates with primer combi nation A02-T02 (both having an additional C at their 3' end) generated auto radiographic AFLP fingerprints that were grouped by numerical analysis in t wo main AFLP clusters allowing clear separation of M. ulcerans (cluster I) from the M. tuberculosis complex members M, bovis and M. tuberculosis (clus ter D), Calculation of similarities using the band-based Dice correlation c oefficient instead of the Pearson product-moment correlation coefficient re vealed a further subgrouping in cluster II. The two resulting subclusters c orresponded with the phenotypic identity of M. bovis and M, tuberculosis, r espectively, and could also be visually identified by two AFLP marker bands . Because of the relatively low degree of genotypic variation among the AFL P band patterns of the latter two taxa, no correlation could be found with previously reported molecular typing data or with geographical origin, The use of primer combination A02-T01 (the latter having an A as selective base ) did not increase the resolving power within the M. tuberculosis complex b ut resulted in a visual subgrouping of the iii ulcerans strains that was no t observed with primer combination A02-T02, Based on the presence or absenc e of a single AFLP marker band, the M ulcerans isolates could be unambiguou sly classified in two continental types corresponding with the African and Australian origin of the strains, respectively. In conclusion, the radioact ive AFLP method proved to be a reproducible and reliable taxonomic tool for the differentiation of the three mycobacterial species under study and als o demonstrated its potential use for typing of M. ulcerans strains when emp loying multiple primer combinations.