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Journal of Clinical Microbiology, October 2000, p. 3675-3680, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

G. Huys,1 L. Rigouts,2 K. Chemlal,2 F. Portaels,2 and J. Swings1,3,*

Laboratory of Microbiology1 and BCCMTM/LMG Culture Collection,3 University Ghent, B-9000 Ghent, and Department of Microbiology, Division of Mycobacteriology, Institute of Tropical Medicine, B-2000 Antwerp,2 Belgium

Received 19 May 2000/Returned for modification 29 June 2000/Accepted 22 July 2000

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.

* Corresponding author. Mailing address: Laboratorium voor Microbiologie, Universiteit Gent, K.L. Ledeganckstr. 35, B-9000 Ghent, Belgium. Phone: 32 9 2645116. Fax: 32 9 2645092. E-mail: jean.swings{at}rug.ac.be.

Journal of Clinical Microbiology, October 2000, p. 3675-3680, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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