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

Abstract

Mycobacterium ulcerans and M. marinum are emerging necrotizing mycobacterial pathogens that reside in common reservoirs of infection and exhibit striking pathophysiological similarities. Furthermore, the interspecific 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, W. M. Meyers, J. Swings, and F. Portaels, Am. J. Trop. Med. Hyg. 64:270-273, 2001). AFLP analysis resulted in profiles which grouped M. ulcerans and M. marinum into two separate clusters. The numerical analysis also revealed subgroups among the M. marinum and M. ulcerans isolates. In conclusion, PRPA appears to provide a rapid method for differentiating the African M. ulcerans type from other geographical types but is unsuitable for interspecific differentiation of M. marinum and M. ulcerans. In comparison, whole- genome techniques such as IS 2404-RFLP and AFLP appear to be far more useful in discriminating between M. marinum and M. ulcerans, and may thus be promising molecular tools for the differential diagnosis of infections caused by these two species.

FIG. 1

Examples of PCR restriction profiles obtained from a representative set of strains using three restriction enzymes, RsaI, DraI, and EcoNI. The first and last lanes show the 100-bp ladder. ND, no digested PCR product; R, D, and E, RsaI, DraI, and EcoNI, respectively, P.N.G., Papua New Guinea.

FIG. 2

A representative Southern blot obtained with 10 M. ulcerans (lanes 1 to 10) and 7 M. marinum (lanes 11 to 17) strains from different geographic origins. Lanes: 1 to 3, African; 4, reference strain ATCC 19423; 5, Australian; 6, Southeast Asian; 7, Asian; 8 and 9, South America; 10, Mexican; 11 and 12, United States; 13, reference strain ATCC 927; 14 and 15, Belgian; lanes 16 and 17, South African. The molecular size (in kilobases) is shown on the left.

FIG. 3

Numerical analysis of normalized AFLP band patterns generated from M. ulcerans (n = 29) and M. marinum (n = 28) using primer combination A02 and T02. In addition, six outlying strains representing other mycobacterial taxa were included: M. tuberculosis (ITM 8004^T), M. bovis (ITM 96-1644), M. africanum (ITM 98-0703), M. kansaii (TON T65/83), and two Mycobacterium strains (TON T31/81 and ITM 98-209). The dendrogram was constructed using the unweighted paired-group using arithmetic averages with correlation levels expressed as percentages of the Pearson product-moment correlation coefficient. The clusters representing M. ulcerans and M. marinum were defined at a delineation level of 60%.

FIG. 4

Hypothetical presentation showing some differential characteristics of M. marinum and M. ulcerans and the postulated position of putative transitory forms between these two taxa.