Genomic Diversity among Drug Sensitive and Multidrug Resistant Isolates of Mycobacterium tuberculosis with Identical DNA Fingerprints

Abstract

Background Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB), is characterized by low sequence diversity making this bacterium one of the classical examples of a genetically monomorphic pathogen. Because of this limited DNA sequence variation, routine genotyping of clinical MTBC isolates for epidemiological purposes relies on highly discriminatory DNA fingerprinting methods based on mobile and repetitive genetic elements. According to the standard view, isolates exhibiting the same fingerprinting pattern are considered direct progeny of the same bacterial clone, and most likely reflect ongoing transmission or disease relapse within individual patients.Methodology/Principal FindingsHere we further investigated this assumption and used massively parallel whole-genome sequencing to compare one drug-susceptible (K-1) and one multidrug resistant (MDR) isolate (K-2) of a rapidly spreading M. tuberculosis Beijing genotype clone from a high incidence region (Karakalpakstan, Uzbekistan). Both isolates shared the same IS6110 RFLP pattern and the same allele at 23 out of 24 MIRU-VNTR loci.We generated 23.9 million (K-1) and 33.0 million (K-2) paired 50 bp purity filtered reads corresponding to a mean coverage of 483.5 fold and 656.1 fold respectively. Compared with the laboratory strain H37Rv both Beijing isolates shared 1,209 SNPs. The two Beijing isolates differed by 130 SNPs and one large deletion. The susceptible isolate had 55 specific SNPs, while the MDR variant had 75 specific SNPs, including the five known resistance-conferring mutations.ConclusionsOur results suggest that M. tuberculosis isolates exhibiting identical DNA fingerprinting patterns can harbour substantial genomic diversity. Because this heterogeneity is not captured by traditional genotyping of MTBC, some aspects of the transmission dynamics of tuberculosis could be missed or misinterpreted. Furthermore, a valid differentiation between disease relapse and exogenous reinfection might be impossible using standard genotyping tools if the overall diversity of circulating clones is limited. These findings have important implications for clinical trials of new anti-tuberculosis drugs.

Whole-Genome-Based Mycobacterium tuberculosis Surveillance: a Standardized, Portable, and Expandable Approach

“…Such large SNP differences among isolates with identical IS6110 patterns in a low-incidence setting is rather unexpected, as this has been considered a reliable marker for isolates belonging to one transmission chain (18). In this regard, we extend similar findings we made previously for two Beijing lineage isolates from an Eastern European high-incidence setting (19) to Haarlem lineage isolates from a Western European low-incidence setting, suggesting that classical genotyping is overestimating rates of recent transmission in general.…”

Evaluation of Two Molecular Assays for Rapid Detection of Mycobacterium tuberculosis Resistance to Fluoroquinolones in High-Tuberculosis and -Multidrug-Resistance Settings

“…Associations between this genotype family and resistance to anti-TB drugs, including RIF, INH, and FQs, have been reported previously (9,10,25,(27)(28)(29)(30)43). In our study only two strains belonged to the T1 family strains, and both of them had the mutation D94N detected in the Beijing family strains as well.…”

Combination of Single Nucleotide Polymorphism and Variable-Number Tandem Repeats for Genotyping a Homogenous Population of Mycobacterium tuberculosis Beijing Strains in China

“…One way to address this challenge is whole-genome sequencing (WGS), which provides the highest discrimination power and where homoplasy is not an issue (7). In recent years, with the development of high-throughput next-generation sequencing technology, several studies have applied WGS to reconstruct transmission chains between VNTR-defined cluster cases (15,22,40), and some clustered strains defined by traditional molecular genotyping methods were identified as distantly related isolates (37). However, because of the relatively high cost and the complexity of data analysis, the application of this technology to study the molecular epidemiology of M.tuberculosis will be limited for some time, especially in developing countries.…”