Multilocus variable-number tandem repeat typing of Mycobacterium ulcerans

Abstract

The apparent genetic homogeneity of Mycobacterium ulcerans contributes to the poorly understood epidemiology of M. ulcerans infection. Here, we report the identification of variable number tandem repeat (VNTR) sequences as novel polymorphic elements in the genome of this species. A total of 19 potential VNTR loci identified in the closely related M. marinum genome sequence were screened in a collection of 23 M. ulcerans isolates, one Mycobacterium species referred to here as an intermediate species, and five M. marinum strains. Nine of the 19 loci were polymorphic in the three species (including the intermediate species) and revealed eight M. ulcerans and five M. marinum genotypes. The results from the VNTR analysis corroborated the genetic relationships of M. ulcerans isolates from various geographical origins, as defined by independent molecular markers. Although these results further highlight the extremely high clonal homogeneity within certain geographic regions, we report for the first time the discrimination of the two South American strains from Surinam and French Guyana. These findings support the potential of a VNTR-based genotyping method for strain discrimination within M. ulcerans and M. marinum.

FIG. 1.

Dendrogram showing the genetic relationships of the M. marinum and M. ulcerans isolates determined using nine VNTR loci. The dendrogram was built using the neighbor-joining algorithm and ITM 1717 and 7732 as outgroups, as described in Material and Methods. The VNTR amplicon sizes and the corresponding genotype designations are indicated at the right. The linkage distance scale and the VNTR locus numbers are indicated at the bottom. MM, M. marinum; MU, M. ulcerans; MI, intermediate isolate.

FIG. 2.

PCR analysis of VNTR locus 19. (A) M. ulcerans and the intermediate strain. Lanes: L, 100-bp DNA ladder; 1, ITM 5142; 2, ITM 5147; 3, ITM 94-1324; 4, ITM 9537; 5, ITM 94-1331; 6, ITM 94-1328; 7, ITM 98-912; 8, ITM 8756; 9, ITM 842; 10, ITM 7922; 11, ITM 5114; 12, ITM 5143; 13, ITM 00-1026; 14, 50-bp ladder (locus 19); 15, ITM 96-658; 16, ITM 97-111; 17, ITM 97-483; 18, ITM 97-104; 19, ITM 5152; 20, ITM 5155; 21, ITM 97-610; 22, ITM 97-648; 23, ITM 97-680; 24, ITM 94-662; 25, ITM 94-511; 26, negative control. (B) M. marinum. Lanes: L, 100-bp DNA ladder; 27, ITM 8022; 28, ITM 1717; 29, ITM 94-56; 30, ITM 1320; 31, ITM 7732; 32, negative control.