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

Abstract

The usefulness of amplified fragment length polymorphism (AFLP) analysis was evaluated for the discrimination of Mycobacterium bovis (17 strains), M. tuberculosis (15 strains), and M. 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 of ApaI/TaqI 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 of M. ulcerans (cluster I) from the M. tuberculosis complex members M. bovis and M. 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 of M. bovis and M. 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 the M. tuberculosis complex but resulted in a visual subgrouping of the M. ulcerans strains that was not observed with primer combination A02-T02. Based on the presence or absence of a single AFLP marker band, the M. 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 of M. ulcerans strains when employing multiple primer combinations.

FIG. 1

Numerical analysis of normalized AFLP band patterns generated from M. bovis (M. bov.) (n = 17), M. tuberculosis (M. tub.) (n = 15), and a subset of M. ulcerans (M. ulc.) strains (n = 3) using primer combination A02-T02. The dendrogram was constructed using the unweighted pair-group method using arithmetic averages with correlation levels expressed as percentage values of the band-based Dice coefficient. M1 and M2 denote species-specific AFLP marker bands differentiating M. tuberculosis from M. bovis. Footnotes: a, data from Rigouts et al. (L. Rigouts, M. Desmecht, J. Dufey, C. Saegerman, K. Kremer, H. Traore, D. van Soolingen, K. Walravens, J. Godfroid, and F. Portaels, submitted for publication); b, data from Portaels et al. (27); c, data not published (all these isolates showed different IS6110-RFLP profiles).

FIG. 2

Normalized AFLP band patterns generated from 12 M. ulcerans strains and one M. tuberculosis reference strain using primer combination A02-T01. M3 denotes a continent-specific AFLP marker band.