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The usefulness of amplified fragment length polymorphism (AFLP) analysis was evaluated for the discrimination ofMycobacterium bovis (17 strains),M. tuberculosis (15 strains), andM. ulcerans (12 strains) at the inter- and intraspecific 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 subsequent ligation of double-stranded restriction site-specific adapter oligonucleotides. Selective amplification ofApa I/Taq I templates with primer combination A02-T02 (both having an additional C at their 3′ end) generated autoradiographic AFLP fingerprints that were grouped by numerical analysis in two main AFLP clusters allowing clear separation ofM. ulcerans (cluster I) from theM. tuberculosis complex membersM. bovis andM. tuberculosis (cluster II). Calculation of similarities using the band-based Dice correlation coefficient instead of the Pearson product-moment correlation coefficient revealed a further subgrouping in cluster II. The two resulting subclusters corresponded with the phenotypic identity ofM. bovis andM. tuberculosis , respectively, and could also be visually identified by two AFLP marker bands. Because of the relatively low degree of genotypic variation among the AFLP 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 theM. tuberculosis complex but resulted in a visual subgrouping of theM. ulcerans strains that was not observed with primer combination A02-T02. Based on the presence or absence of a single AFLP marker band, theM. ulcerans isolates could be unambiguously classified in two continental types corresponding with the African and Australian origin of the strains, respectively. In conclusion, the radioactive AFLP method proved to be a reproducible and reliable taxonomic tool for the differentiation of the three mycobacterial species under study and also demonstrated its potential use for typing ofM. ulcerans strains when employing multiple primer combinations.

journal of clinical microbiology

Evaluation of Amplified Fragment Length Polymorphism Analysis for Inter- and Intraspecific Differentiation of Mycobacterium bovis , M. tuberculosis , and M. ulcerans