Research Article: A Single-Step Sequencing Method for the Identification of Mycobacterium tuberculosis Complex Species

Date Published: June 18, 2008

Publisher: Public Library of Science

Author(s): Zoheira Djelouadji, Didier Raoult, Mamadou Daffé, Michel Drancourt, Pamela L. C. Small

Abstract: BackgroundThe Mycobacterium tuberculosis complex (MTC) comprises closely related species responsible for strictly human and zoonotic tuberculosis. Accurate species determination is useful for the identification of outbreaks and epidemiological links. Mycobacterium africanum and Mycobacterium canettii are typically restricted to Africa and M. bovis is a re-emerging pathogen. Identification of these species is difficult and expensive.Methodology/Principal FindingsThe Exact Tandem Repeat D (ETR-D; alias Mycobacterial Interspersed Repetitive Unit 4) was sequenced in MTC species type strains and 110 clinical isolates, in parallel to reference polyphasic identification based on phenotype profiling and sequencing of pncA, oxyR, hsp65, gyrB genes and the major polymorphism tandem repeat. Inclusion of M. tuberculosis isolates in the expanding, antibiotic-resistant Beijing clone was determined by Rv0927c gene sequencing. The ETR-D (780-bp) sequence unambiguously identified MTC species type strain except M. pinnipedii and M. microti thanks to six single nucleotide polymorphisms, variable numbers (1–7 copies) of the tandem repeat and two deletions/insertions. The ETR-D sequencing agreed with phenotypic identification in 107/110 clinical isolates and with reference polyphasic molecular identification in all isolates, comprising 98 M. tuberculosis, 5 M. bovis BCG type, 5 M. canettii, and 2 M. africanum. For M. tuberculosis isolates, the ETR-D sequence was not significantly associated with the Beijing clone.Conclusions/SignificanceETR-D sequencing allowed accurate, single-step identification of the MTC at the species level. It circumvented the current expensive, time-consuming polyphasic approach. It could be used to depict epidemiology of zoonotic and human tuberculosis, especially in African countries where several MTC species are emerging.

Partial Text: The Mycobacterium tuberculosis complex (MTC) comprises several closely related species responsible for strictly human and zoonotic tuberculosis (Figure 1). In addition to M. tuberculosis, which represents the leading cause of human tuberculosis worldwide and is now emerging as extensively drug-resistant tuberculosis strains [1], other MTC species have been found in patients, typically in African countries (Figure 2). Mycobacterium bovis is a re-emerging, zoonotic agent of bovine tuberculosis [2] whose prevalence probably depends on variations in direct exposure to cattle and consumption of unpasteurised dairy products [3]. The prevalence of Mycobacterium africanum type I (West Africa) and type II (East Africa) [4] has decreased in several African countries over the last decades [5],[6]. Mycobacterium canettii, a rare MTC species, has been isolated recently in patients exposed in Africa [7]. Mycobacterium microti, a vole and small rodent pathogen [8] that is closely related to the so-called Dassie-bacillus and infects small mammals in South Africa and the Middle East [9],[10], has been isolated in humans [11]. Mycobacterium caprae is a rare cause of tuberculosis in cattle [12],[13] and zoonotic tuberculosis in humans [14] while Mycobacterium pinnipedii has been isolated from seal lions and fur seals [15]. A recent description of the re-emergence of M. bovis in cattle, along with the direct interhuman transmission of this zoonotic organism [16] in a six-case cluster that included one death in United Kingdom [17], illustrates the potential of emerging and re-emerging zoonotic tuberculosis due to MTC species other than M. tuberculosis and the necessity for accurate species identification.

Previous methods for MTC species identification either combined the amplification of several genomic regions in order to identify all species [27],[48] or analyzed one gene polymorphism to distinguish between only two species. ETR-D spacer sequencing herein developed proved to be specific for the MTC and allowed the differentiation of the 7/8 MTC species in a single reaction. Indeed all the non-tuberculosis mycobacteria yielded a negative ETR-D PCR amplification as previously described [49].

Source:

http://doi.org/10.1371/journal.pntd.0000253

 

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