Characterising virulence and AMR in Mycobacterium tuberculosis

There are over 10 million new cases of TB per year, 600,000 of which were caused by multidrug‑resistant (MDR) strains of Mycobacterium tuberculosis that are resistant to the first‑line drugs of rifampicin and isoniazid¹.

More worrying still is the increase in extensively drug‑resistant (XDR) strains that exhibit additional resistance to other classes of drugs. The mechanisms underlying the development of highly transmissible XDR strains are not fully elucidated and studies using short‑ read sequencing technology have limited capacity to resolve the structural variations, gene duplications and repetitive regions which may contribute to resistance, virulence and transmission. To combat these challenges, researchers at The University of Queensland, Australia, are utilising the long sequencing reads provided by nanopore technology to provide a more comprehensive understanding of the evolutionary mechanisms underlying the emergence of highly transmissible strains of M. tuberculosis².

The team performed whole genome shotgun sequencing of an XDR strain of M. tuberculosis causative of drug resistant outbreaks in the Western Province of Papua New Guinea. Using the assembly tool CANU³ with sequence polishing using Racon⁴, a complete circular  de novo assembly was generated with an average read depth of 273x and a base accuracy of 99.95%.

Analysis of the genome allowed the drug resistance profile to be determined with complete phenotypic concordance.

It was also possible to identify all relevant drug resistance conferring single nucleotide polymorphisms. Further, the team identified mutations in efflux pump genes, transporters, cell wall biosynthesis genes and virulence genes that may contribute to the drug resistance phenotype and successful transmission of this strain. Of particular note was the accurate characterisation of the GC rich and highly repetitive proline‑glutamate/proline‑ proline‑glutamate (PE/PPE) genes. These genes are implicated in immune invasion and virulence and are of increasing interest in TB research.

Many of the genes studied were not present in a genome assembly created using short‑read sequencing technology, which the researchers suggest is due to their high GC content and, in some cases, deletions which disrupt the open reading frame. Comparison of the genome with those of other lineages of M. tuberculosis, showed 2 regions (~4.5 kb and 390 bp) that were absent in Euro‑American lineage strains (Figure 11).

Summarising their research, the team stated:

"…the assembly of a complete genome of a XDR “epidemic strain”  using nanopore technology did not only provide proof of principle for future deployment of this technology in settings endemic for drug resistant TB but it also demonstrated the use of this technology in further understanding of M. tuberculosis genetics. It characterised the drug resistance profile and potential virulence factors found in this strain, and provided a reference strain for future genome assembly and mapping"².

This case study is taken from the Microbiology white paper.