Characterization of Microevolution Events in Mycobacterium tuberculosis Strains Involved in Recent Transmission Clusters

Abstract

Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variable-number tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1-to 5-year period. The IS6110 insertion sites from 16 representative isolates of reference and microevolved variants were mapped by ligation-mediated PCR in order to characterize the genetic background involved in microevolution. Both intragenic and intergenic IS6110 locations resulted from these microevolution events. Among those cases of IS6110 locations in intergenic regions which could have an effect on the regulation of adjacent genes, we identified the overexpression of cytochrome P450 in one microevolved variant using quantitative real-time reverse transcription-PCR. Our results help to define the frequency with which microevolution can be expected in M. tuberculosis transmission chains. They provide a snapshot of the genetic background of these subtle rearrangements and identify an event in which IS6110-mediated microevolution in an isogenic background has functional consequences.Mycobacterium tuberculosis is characterized by high genetic homogeneity (99.9% similarity at the nucleotide level) (40). Different mechanisms are involved in the acquisition of variability in M. tuberculosis and include single-nucleotide polymorphisms, insertions, deletions, genomic rearrangements, and transpositions (23). One of the mobile genetic elements involved in transposition events, the insertion sequence IS6110 (22, 42), is considered a key mechanism in the evolution of M. tuberculosis.IS6110 transposition events are responsible not only for the specific genomic changes directly caused by insertion sequence mobility. Extensive chromosomal rearrangements involving large deletions by IS6110-mediated homologous recombination have also been described (10), and their entry can modify the expression profiles of adjacent genes (32).IS6110 has been used extensively as a genotypic marker in epidemiological studies. Application of M. tuberculosis fingerprinting based on IS6110 restriction fragment length polymorphism (RFLP) has allowed us to refine the identification of recent transmission events. The M. tuberculosis isolates of cases involved in a recent transmission chain generally have identical fingerprints and thus constitute a cluster. However, ...

In Vivo IS 6110 Profile Changes in a Mycobacterium tuberculosis Strain as Determined by Tracking over 14 Years

“…Concerning this, most IS6110 copies have been located outside the DnaA boxes, and the oriC activity has not been affected (26). In this sense, the insertion of IS6110 in the oriC region could be an evolutionary advantage (7). In our study, we observed that the insertion of IS6110 is located outside the described DnaA boxes; therefore, we can assume that IS6110 does not decrease the activity of the oriC region.…”

“…Minor variations in IS6110 fingerprints, spoligotypes, or mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) loci reveal microevolution in clonal infections (6,8). These changes could have effects on the molecular structure of the mycobacterial genome and the biology of the bacillus (7).…”

“…The IS6110 element itself modulates expression of neighboring genes and, depending on its location, could confer both transmission ability and virulence (3)(4)(5). Microevolution events due to the transposition of IS6110 are usually reflected in variations of restriction fragment length polymorphism (RFLP) patterns (6,7). Minor variations in IS6110 fingerprints, spoligotypes, or mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) loci reveal microevolution in clonal infections (6,8).…”

Study of Mycobacterium Tuberculosis by Molecular Methods in Northeast Mexico

“…Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variablenumber tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1-to 5-year period (Pérez-Lago et al, 2011), another study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India (Varma-Basil et al 2011) and finally, a study where Forty three isoniazid (INH)-resistant M. tuberculosis isolates were characterized on the basis of the most common INH associated mutations, katG315 and mabA -15C→T, and phenotypic properties (i.e. MIC of INH, resistance associated pattern, and catalase activity).…”

The role of Azeri patients in transmission of tuberculosis to East Azerbaijan, Iran

“…6 The IS6110 is an insertion sequence that belongs to IS3 family. IS6110 commonly used for detection of MTB, 7 epidemiological studies 8 and recent transmission. 9 In IS6110-restriction …”