Evolution and clonal traits of Mycobacterium tuberculosis complex in Guinea-Bissau

Abstract

Two hundred twenty-nine consecutive isolates of Mycobacterium tuberculosis complex from patients with pulmonary tuberculosis in Guinea-Bissau, which is located in West Africa, were analyzed for clonal origin by biochemical typing and DNA fingerprinting. By using four biochemical tests (resistance to thiophene-2-carboxylic acid hydrazide, niacin production, nitrate reductase test, and pyrazinamidase test), the isolates could be assigned to five different biovars. The characteristics of four strains conformed fully with the biochemical criteria for M. bovis, while those of 85 isolates agreed with the biochemical criteria for M. tuberculosis. The remaining 140 isolates could be allocated into one of three biovars (biovars 2 to 4) representing a spectrum between the classical bovine (biovar 1) and human (biovar 5) tubercle bacilli. By using two genotyping methods, restriction fragment length polymorphism analysis with IS6110 (IS6110 RFLP analysis) and spoligotyping, the isolates could be separated into three groups (groups A to C) of the M. tuberculosis complex. Group A (n = 95), which contained the majority of classical human M. tuberculosis isolates, had large numbers of copies of IS6110 elements (mean number of copies, 9) and a distinctive spoligotyping pattern that lacked spacers 33 to 36. Isolates of the major group, group B (n = 119), had fewer IS6110 copies (mean copy number, 5) and a spoligotyping pattern that lacked spacers 7 to 9 and 39 and mainly comprised isolates of biovars 1 to 4. Group C isolates (n = 15) had one to three IS6110 copies, had a spoligotyping pattern that lacked spacers 29 to 34, and represented biovar 3 to 5 isolates. Four isolates whose biochemical characteristics conformed with those of M. bovis clustered with the group B isolates and had spoligotype patterns that differed from those previously reported for M. bovis, in that they possessed spacers 40 to 43. Interestingly, isolates of group B and, to a certain extent, also isolates of group C showed a high degree of variability in biochemical traits, despite genotypic identity in terms of IS6110 RFLP and spoligotype patterns. We hypothesize that isolates of groups B and C have their evolutionary origin in West Africa, while group A isolates are of European descent.

FIG. 1

IS6110 banding patterns and similarity matrices for 229 M. tuberculosis complex isolates from Guinea-Bissau. Banding patterns are ordered by similarity. The corresponding dendrograms are to the left of the panels. The positions of the bands in each lane are adjusted (normalized) so that band positions for all strains are comparable. Scale depicts similarity coefficients (which are defined elsewhere [29]). Groups A to C are as defined in the Results.

FIG. 2

Number of IS6110 copies present in isolates of five different biovars of the M. tuberculosis complex. Differences in numbers of IS6110 copies between biovar 5 (classical human M. tuberculosis) and biovars 2, 3, and 4 were significant (P < 0.01 by the Wilcoxon rank sum test).

FIG. 3

Schematic representation of spoligotypes of 32 M. tuberculosis isolates belonging to cluster B:5. The isolates are sorted in the same order in which they were sorted by their IS6110 RFLP patterns in Fig. 1. X, positive hybridization signals; dots, lack of hybridization.