Genomic Analysis of Mycobacterium tuberculosis Isolates and Construction of a Beijing Lineage Reference Genome

Abstract

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, kills over 1 million people worldwide annually. Development of drug resistance (DR) in the pathogen is a major challenge for TB control. We conducted whole-genome analysis of seven Taiwan M. tuberculosis isolates: One drug susceptible (DS) and five DR Beijing lineage isolates and one DR Euro-American lineage isolate. Developing a new method for DR mutation identification and applying it to the next-generation sequencing (NGS) data from the 6 Beijing lineage isolates, we identified 13 known and 6 candidate DR mutations and provided experimental support for 4 of them. We assembled the genomes of one DS and two DR Beijing lineage isolates and the Euro-American lineage isolate using NGS data. Moreover, using both PacBio and NGS sequencing data, we obtained a high-quality assembly of an extensive DR Beijing lineage isolate. Comparative analysis of these five newly assembled genomes and two published complete genomes revealed a large number of genetic changes, including gene gains and losses, indels and translocations, suggesting rapid evolution of M. tuberculosis. We found the MazEF toxin–antitoxin system in all the seven isolates studied and several interesting mutations in MazEF proteins. Finally, we used the four assembled Beijing lineage genomes to construct a high-quality Beijing lineage reference genome that is DS and contains all the genes in the four genomes. It contains 212 genes not found in the standard reference H37Rv, which is Euro-American. It is therefore a better reference than H37Rv for the Beijing lineage, the predominant lineage in Asia.

Large-scale Pan Genomic Analysis of Mycobacterium tuberculosis Reveals Key Insights Into Molecular Evolutionary Rate of Specific Processes and Functions

“…For example, comparative analysis of the genomes of drug susceptible and drug resistant Mtb isolates suggests rapid evolution of Mtb. 8 In addition, within-host microevolution of Mtb is characterised by elevated rates of nucleotide substitution which have implications for the appearance of AR. 5 On a larger scale, high levels of mutation resulting in significant genomic diversity and the rapid evolution of Mtb have consistently been identified in different regions of the world shedding light on the emergence of AR.…”

“… 5 On a larger scale, high levels of mutation resulting in significant genomic diversity and the rapid evolution of Mtb have consistently been identified in different regions of the world shedding light on the emergence of AR. 3 , 4 , 8 - 11 Among the numerous Mtb virulence factors, PE/PPE genes have been shown to evolve at high rates due to particularly high frequency of mutations. 12 , 13 At the same time, varying rates of mutation or evolution of Mtb have been demonstrated to occur during human infection, with decreasing mutation rates through latent infection 14 and high mutation rates during active disease.…”

Connection between two historical tuberculosis outbreak sites in Japan, Honshu, due to a new ancestral L2 sublineage

“…The "Modern" Beijing represents the clade that is at the end of the phylogenetic tree and it encompasses at least 7 large and epidemiologically acknowledged sublineages (Asian African 1, Asian African 3, Europe/Russia B0/W148 outbreak, Central Asia, Asian African 2, Asian African2/RD142, Pacific RD150) [31]. Interestingly, apart from the SNPs discovered these recent years, other major genomic rearrangements were shown to have happened in some L2 genomes [33,34]. Modern Beijing strains are responsible of most but not of all of the recent MDR-TB outbreaks and are more transmissible [4,35,36].…”