Evaluation of PCR-Restriction Profile Analysis and IS 2404 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 Mycobacterium ulcerans and M. marinum are slow-growing mycobacterial species with optimal growth temperatures of 30 to 33°C. These organisms are emerging as clinically significant pathogens associated with skin infections (5, 9). M. ulcerans infection, or Buruli ulcer (BU), was first described in Bairnsdale, Australia, in 1948 (17) and was subsequently found in numerous, mostly tropical countries in Africa, the Americas, Southeast Asia, and the central Pacific. Recent reports describe increases in the incidence of BU in Benin (13), Australia (6,8,12,34), and Côte d'Ivoire (18). M. ulcerans causes chronic necrotizing ulcers in the skin of humans (22) and other mammals (22, 23). The epidemiology of BU is poorly understood, but most foci are associated with slow-flowing or stagnant water; however, the natural reservoir of M. ulcerans remains unknown. M. marinum, first described in Sweden (1), gives rise to infections in temperate climates and is the cause of fish tank and swimming pool granulomas (16).M. ulcerans is often difficult to isolate from clinical specimens and usually requires 6 to 8 weeks to produce visible growth in primary culture (23,24). Definitive identification of M. ulcerans is thus time-consuming; however, it can be recognized by classic molecular and microbiologic methods (20,24). M. marinum, once cultured, is readily identified by using conventional mycobacterial characterization methods. It grows relatively quickly (1 to 2 weeks) and is easily recognized as a result of its photochromogenicity (20). While infections due to M. marinum can usually be treated with antimycobacterial drugs, very few cases of BU lesions respond favorably to antimicrobial therapy (2), making wide surgical excision and skin grafting the treatment of choice.In the last decade, various DNA-based techniques have been used to classify mycobacteria (15,25,26,30). All such studies have demonstrated a high taxonomic affiliation between M. ulcerans and M. marinum. Other attempts have targeted the 3Ј end of 16S rRNA gene and found four subtypes of M. ulcerans related to their geographic origin, except for one isolate from Suriname, which exhibited the same sequence as M. marinum (20). The use of IS2404 resriction fragment length polymorphism (RFLP) analysis (2) led to the classification...

Mycobacterium marinum Infections in Fish and Humans in Israel

“…Also, with this method the resolution (18,24,25), these two species were distinguished by AFLP at 60% similarity. Our results are in agreement with those of Chemlal et al (4). M. marinum infections in Thailand are reported as being frequent among fishermen and fishmongers (13).…”

Characterization of an Unusual Mycobacterium: a Possible Missing Link between Mycobacterium marinum and Mycobacterium ulcerans

“…The pattern developed from these findings was confirmed by various DNA-based techniques, which demonstrated a strong taxonomic relationship between these two organisms (11,24,25,33,39). However, further analysis of the genomes of these two closely related species has revealed differences (4,46,49). Next to the separation of representatives of both species into two clusters by the amplified fragment length polymorphism (AFLP) analysis technique (4,42), the finding that most clearly discriminates the two species is the presence of insertion sequence IS2404 in M. ulcerans in high copy numbers but a total absence of IS2404 in all M. marinum isolates from different geographic regions in-vestigated (4,46).…”

“…. .indicating an evolving characteristic within the taxon" (4). In the present study, we determined the phenotypic and genotypic profiles of an isolate, designated ITM 00-1026 (IPP 2000-372), from a French patient.…”

GenotypingMycobacterium ulceransandMycobacterium marinumby Using Mycobacterial Interspersed Repetitive Units

“…PCR-restriction profile analyses of 16S rRNAs showed four different genotypic profiles of M. ulcerans, i.e., the African, Southeast Asian, Mexican, and South American profiles (3). Amplified fragment length polymorphism clearly separates M. ulcerans from M. marinum but has limited use for intraspecies differentiation (3). Sequencing of the 3Ј end of the 16S rRNA (30) categorized M. ulcerans into the following five types based on the continent of origin: one African type, one Australian type, one Mexican type, one Asian type, and one South American type.…”

“…PCR-restriction profile analyses of 16S rRNAs showed four different genotypic profiles of M. ulcerans, i.e., the African, Southeast Asian, Mexican, and South American profiles (3). Amplified fragment length polymorphism clearly separates M. ulcerans from M. marinum but has limited use for intraspecies differentiation (3).…”