Mycobacterium africanumGenotyping UsingNovel Spacer Oligonucleotides in the Direct RepeatLocus

Abstract

This study involves a first evaluation of 25 novel spacer oligonucleotides in addition to the 43 routine spacers for molecular characterization of a panel of 65 isolates of tubercle bacilli from different geographic origins that were initially classified as Mycobacterium africanum based on phenotypic characters. The 68-spacer format defined four additional patterns, and three groups were identified. The relatively homogeneous groups A1 and A2 included strains from West Africa, and A3-1 included strains from East Africa. The presence of deletion region RD9 confirmed the reclassification of the M. africanum subtype II spoligopattern within group A3-1 as Mycobacterium tuberculosis. These isolates may represent a diverging branch of M. tuberculosis in Africa. The use of new spacers also suggested an undergoing evolution of M. africanum subtype I in West Africa. Our results showed that the strain differentiation within the M. tuberculosis complex is improved by using novel spacers, and extensive studies using new-generation spoligotyping may be helpful to better understand the evolution of M. africanum.Many recent studies have used spoligotyping, a commonly used PCR-based method, for Mycobacterium tuberculosis complex identification and genotyping (13,19). Recently, VianaNiero et al. showed that a collection of isolates tentatively identified as Mycobacterium africanum could be successfully subdivided into three groups, A1 to A3, by spoligotyping, IS6110-restriction fragment length polymorphism analysis, and analysis by variable number of tandem DNA repeats and that a specific spoligotyping signature of M. africanum was the absence of spacers 8, 9, and 39 (25). This was followed by a study on isolates from Uganda, which showed that M. africanum subtype II differed from the classical M. africanum subtype I isolates by the absence of spacers 33 to 36 and 40 (16, 21).Although spoligotyping is easier to perform than IS6110-restriction fragment length polymorphism analysis, its discriminatory power is not optimal when it is used in a routine 43-spacer format (14). Recently, a panel of novel spacers was used in spoligotyping to improve M. tuberculosis complex differentiation (23, 24). As these studies did not investigate any M. africanum strains, we thought it desirable to investigate the variability of a set of novel sequences in addition to the routine spoligotyping spacers with a panel of isolates of tubercle bacilli from different geographic origins that were initially classified as M. africanum based on phenotypic characters. The present study involves a first evaluation of the 25 novel spoligotyping spacers for M. africanum characterization. MATERIALS AND METHODSMycobacterial strains. This study used a collection of 65 clinical isolates from Benin, Cameroon, France, Guinea-Bissau, Nigeria, Senegal, Sierra-Leone, and Uganda and from African patients residing in the United States. Twenty-nine clinical isolates were from the National Reference Laboratory for Mycobacteria, Institut Pasteur, Paris, France, and 36 were fr...

Molecular Evolutionary History of Tubercle Bacilli Assessed by Study of the Polymorphic Nucleotide within the Nitrate Reductase ( narGHJI ) Operon Promoter

“…Hence, our data support the call to do away with the unnecessary "subtype II/East African/M. tuberculosis-like" M. africanum designation (1, 23) since, in every case reported to date, strains identified as such were later found to really be either M. tuberculosis PGG1 or PGG2 isolates rather than true M. africanum when reevaluated by molecular means (1,2,13,19,20).…”

Discriminatory Power and Reproducibility of Novel DNA Typing Methods forMycobacterium tuberculosisComplex Strains

“…These spacers were found to increase the ability of spoligotyping to discriminate between M. bovis, M. caprae, and M. microti strains and also showed polymorphisms between strains of the M. tuberculosis Beijing genotype (34,71,73). In a recent study by Brudey et al (11) it was shown that the additional spacers enhanced the ability of spoligotyping to differentiate between M. africanum isolates. In the present study we found that the inclusion of these novel spacers did not improve the overall discriminatory power of the method in comparison with the traditional spoligotyping when applied to 90 isolates of mainly M. tuberculosis.…”

“…In the previous study the species were determined on the basis of a combination of biochemical testing and DNA typing (36). However, identification of mycobacterial strains has since improved by the use of chromosomal deletions (10,28,44,48), SNPs (28), and sequencing of the 16S rRNA gene DNA (8,33 (11,44,45). The latter designation was established after revision of the interpretative guidelines used for analysis of genetic data (44).…”

Unexpectedly High Proportion of Ancestral Manu Genotype Mycobacterium tuberculosis Strains Cultured from Tuberculosis Patients in Egypt

“…The present study is a first attempt to provide insight into the population structure of M. tuberculosis in Egypt. We selected spoligotyping to create this snapshot, as it has been shown to be a valuable method for determination of the clonal and phylogenetic relationships of M. tuberculosis strains, providing in parallel useful information about TB in a given population, in a given country, among countries, and throughout the world (4,5,9,10,12,16,17,19 Detailed spoligotyping results are summarized in Tables 2 and 3, while the evolutionary relationships of the strains are illustrated in an MST shown in Fig. 1.…”