Deciphering drug resistance in Mycobacterium tuberculosis using whole-genome sequencing: Progress, promise, and challenges

Tuberculosis (TB) is a global infectious threat that is intensified by an increasing incidence of highly drug-resistant disease. Whole-genome sequencing (WGS) studies of Mycobacterium tuberculosis, the causative agent of TB, have greatly increased our understanding of this pathogen. Since the first M. tuberculosis genome was published in 1998, WGS has provided a more complete account of the genomic features that cause resistance in populations of M. tuberculosis, has helped to fill gaps in our knowledge of how both classical and new antitubercular drugs work, and has identified specific mutations that allow M. tuberculosis to escape the effects of these drugs. WGS studies have also revealed how resistance evolves both within an individual patient and within patient populations, including the important roles of de novo acquisition of resistance and clonal spread. These findings have informed decisions about which drug-resistance mutations should be included on extended diagnostic panels. From its origins as a basic science technique, WGS of M. tuberculosis is becoming part of the modern clinical microbiology laboratory, promising rapid and improved detection of drug resistance, and detailed and real-time epidemiology of TB outbreaks. We review the successes and highlight the challenges that remain in applying WGS to improve the control of drug-resistant TB through monitoring its evolution and spread, and to inform more rapid and effective diagnostic and therapeutic strategies.Pattern Recognition and Bioinformatic

Extensive global movement of multidrug-resistant <em>M. tuberculosis </em>strains revealed by whole-genome analysis

Background: While the international spread of multidrug-resistant (MDR) Mycobacterium tuberculosis strains is an acknowledged public health threat, a broad and more comprehensive examination of the global spread of MDR-tuberculosis (TB) using whole-genome sequencing has not yet been performed. Methods: In a global dataset of 5310 M. tuberculosis whole-genome sequences isolated from five continents, we performed a phylogenetic analysis to identify and characterise clades of MDR-TB with respect to geographic dispersion. Results: Extensive international dissemination of MDR-TB was observed, with identification of 32 migrant MDR-TB clades with descendants isolated in 17 unique countries. Relatively recent movement of strains from both Beijing and non-Beijing lineages indicated successful global spread of varied genetic backgrounds. Migrant MDR-TB clade members shared relatively recent common ancestry, with a median estimate of divergence of 13-27 years. Migrant extensively drug-resistant (XDR)-TB clades were not observed, although development of XDR-TB within migratory MDR-TB clades was common. Conclusions: Application of genomic techniques to investigate global MDR migration patterns revealed extensive global spread of MDR clades between countries of varying TB burden. Further expansion of genomic studies to incorporate isolates from diverse global settings into a single analysis, as well as data sharing platforms that facilitate genomic data sharing across country lines, may allow for future epidemiological analyses to monitor for international transmission of MDR-TB. In addition, efforts to perform routine whole-genome sequencing on all newly identified M. tuberculosis, like in England, will serve to better our understanding of the transmission dynamics of MDR-TB globally.Pattern Recognition and Bioinformatic

SynerClust: A highly scalable, synteny-aware orthologue clustering tool

Accurate orthologue identification is a vital component of bacterial comparative genomic studies, but many popular sequence-similarity-based approaches do not scale well to the large numbers of genomes that are now generated routinely. Furthermore, most approaches do not take gene synteny into account, which is useful information for disentangling paralogues. Here, we present SynerClust, a user-friendly synteny-aware tool based on synergy that can process thousands of genomes. SynerClust was designed to analyse genomes with high levels of local synteny, particularly prokaryotes, which have operon structure. SynerClust’s run-time is optimized by selecting cluster representatives at each node in the phylogeny; thus, avoiding the need for exhaustive pairwise similarity searches. In benchmarking against Roary, Hieranoid2, PanX and Reciprocal Best Hit, SynerClust was able to more completely identify sets of core genes for datasets that included diverse strains, while using substantially less memory, and with scalability comparable to the fastest tools. Due to its scalability, ease of installation and use, and suitability for a variety of computing environments, orthogroup clustering using SynerClust will enable many large-scale prokaryotic comparative genomics efforts.Pattern Recognition and Bioinformatic

Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance

Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.Accepted author manuscriptPattern Recognition and Bioinformatic