Evaluation of PCR-Restriction Profile Analysis and IS2404 Restriction Fragment Length Polymorphism and Amplified Fragment Length Polymorphism Fingerprinting for Identification and Typing of Mycobacterium ulcerans and M. marinum

doi:10.1128/JCM.39.9.3272-3278.2001

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Journal of Clinical Microbiology, September 2001, p. 3272-3278, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3272-3278.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Evaluation of PCR-Restriction Profile Analysis and IS2404 Restriction Fragment Length Polymorphism and Amplified Fragment Length Polymorphism Fingerprinting for Identification and Typing of Mycobacterium ulcerans and M. marinum

K. Chemlal,^*^,¹ G. Huys,² P.-A. Fonteyne,¹ V. Vincent,⁴ A. G. Lopez,¹ L. Rigouts,¹ J. Swings,²^,⁵ W. M. Meyers,³ and F. Portaels¹

Department of Microbiology, Mycobacteriology Unit, Institute of Tropical Medicine, B-2000 Antwerp,¹ and Laboratorium Voor Microbiologie² and BCCM/LMG Culture Collection,⁵ Universiteit Gent, B-9000 Gent, Belgium; Armed Forces Institute of Pathology, Washington, D.C. 20306³; and Laboratoire de Référence des Mycobactéries, Institut Pasteur, 75724 Paris Cedex, France⁴

Received 11 December 2000/Returned for modification 28 March 2001/Accepted 29 May 2001

Mycobacterium ulcerans and M. marinum are emerging necrotizing mycobacterial pathogens that reside in common reservoirs ofinfection and exhibit striking pathophysiological similarities.Furthermore, the interspecific taxonomic relationship betweenthe two species is not clear as a result of the very high phylogeneticrelatedness (i.e., >99.8% 16S rRNA sequence similarity), in contrastto only 25 to 47% DNA relatedness. To help understand the genotypicaffiliation between these two closely related species, we performeda comparative analysis including PCR restriction profile analysis(PRPA), IS2404 restriction fragment length polymorphism (RFLP),and amplified fragment length polymorphism (AFLP) on a set ofM. ulcerans (n = 29) and M. marinum (n = 28) strains recoveredfrom different geographic origins. PRPA was based on a triplerestriction of the 3' end region of 16S rRNA, which differentiatedM. ulcerans into three types; however, the technique could notdistinguish M. marinum from M. ulcerans isolates originating fromSouth America and Southeast Asia. RFLP based on IS2404 producedsix M. ulcerans types related to six geographic regions and didnot produce any band with M. marinum, confirming the previousfindings of Chemlal et al. (K. Chemlal, K. DeRidder, P. A. Fonteyne,W. M. Meyers, J. Swings, and F. Portaels, Am. J. Trop. Med. Hyg.64:270-273, 2001). AFLP analysis resulted in profiles which groupedM. ulcerans and M. marinum into two separate clusters. The numericalanalysis also revealed subgroups among the M. marinum and M. ulceransisolates. In conclusion, PRPA appears to provide a rapid methodfor differentiating the African M. ulcerans type from other geographicaltypes but is unsuitable for interspecific differentiation of M.marinum and M. ulcerans. In comparison, whole- genome techniquessuch as IS 2404-RFLP and AFLP appear to be far more useful indiscriminating between M. marinum and M. ulcerans, and may thusbe promising molecular tools for the differential diagnosis ofinfections caused by these twospecies.

^* Corresponding author. Mailing address: Department of Microbiology, Mycobacteriology Unit, Institute of Tropical Medicine,Nationalestraat 155, B-2000 Antwerp, Belgium. Phone: 32(3)247-63-36.Fax: 32(3)247-63-33. E-mail: kchemlal{at}itg.be.

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