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 are emerging necrotizing mycobacterial pathogens that reside in common reservoirs of infection and exhibit striking pathophysiological similarities. Furthermore, the inter-specific taxonomic relationship between the two species is not clear as a result of the very high phylogenetic relatedness (i.e.,>99.8 % 16S rRNA sequence similarity), in contrast to only 25 to 47 % DNA relatedness. To help understand the genotypic affiliation between these two closely related species, we performed a comparative analysis including PCR restriction profile analysis (PRPA), IS2404 restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) on a set of M. ulcerans (n 29) and M. marinum (n 28) strains recovered from different geographic origins. PRPA was based on a triple restriction of the 3 end region of 16S rRNA, which differentiated M. ulcerans into three types; however, the technique could not distinguish M. marinum from M. ulcerans isolates originating from South America and Southeast Asia. RFLP based on IS2404 produced six M. ulcerans types related to six geographic regions and did not produce any band with M. marinum, confirming the previous findings of Chemlal et al. (K. Chemlal, K. DeRidder, P. A. Fonteyne