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

Mycobacterium ulcerans and M. marinum are emerging necrotizing mycobacteria l pathogens that reside in common reservoirs of infection and exhibit strik ing pathophysiological similarities. Furthermore, the interspecific taxonom ic relationship between the two species is not clear as a result of the ver y high phylogenetic relatedness (i.e., >99.8% 16S rRNA sequence similarity) , in contrast to only 25 to 47% DNA relatedness. To help understand the gen otypic affiliation between these two closely related species, we performed a comparative analysis including PCR restriction profile analysis (PRPA), I S2404 restriction fragment length polymorphism (RFLP), and amplified fragme nt length polymorphism (AFLP) on a set of M. ulcerans (n = 29) and M. marin um (n = 28) strains recovered from different geographic origins. PRPA was b ased on a triple restriction of the 3' end region of 16S rRNA, which differ entiated M. ulcerans into three types; however, the technique could not dis tinguish M. marinum from AL ulcerans isolates originating from South Americ a and Southeast Asia. RFLP based on IS2404 produced six AL ulcerans types r elated to six geographic regions and did not produce any band with M. marin um, confirming the previous findings of Chemlal et al. (K. Chemlal, K. DeRi dder, P. A. Fonteyne, VV. M. Meyers, J. Swing and F. Portaels, Am. J. Trop. Med. Hyg. 64:270-273, 2001). AFLP analysis resulted S, in profiles which g rouped M. ulcerans and M. marinum into two separate clusters. The numerical analysis also revealed subgroups among the M. marinum and M. ulcerans isol ates. In conclusion, PRPA appears to provide a rapid method for differentia ting the African M. ulcerans type from other geographical types but is unsu itable for interspecific differentiation of M. marinum and M. ulcerans. In comparison, whole- genome techniques such as IS 2404-RFLP and AFLP appear t o be far more useful in discriminating between AL Marinum and M. ulcerans, and may thus be promising molecular tools for the differential diagnosis of infections caused by these two species.