Evolution of antimicrobial resistance in Mycobacterium tuberculosis studied in the field and the laboratory

Human tuberculosis (TB), caused by the bacterium M. tuberculosis, is the leading cause of death due to an infectious agent. In 2017, there were an estimate of 10 million new cases and 1.6 million deaths caused by M. tuberculosis. The evolution of drug resistant M. tuberculosis variants is threatening disease control efforts. Generating timely and accurate drug susceptibility testing results is essential for the administration of effective treatment. In M. tuberculosis, drug resistance is primarily conferred by well-defined chromosomally encoded mutations, which offers the possibility of deducing antibiograms by sequencing the drug-resistance related target genes and detecting mutations therein. We assessed the utility of whole genome sequencing in predicting accurate drug susceptibility profiles, as well as predicting quantitative levels of drug resistance in TB.
It has generally been assumed that drug resistant M. tuberculosis variants do not transmit efficiently, as drug resistance comes at a cost. Drug resistance-conferring mutations often target essential, highly conserved genes, causing a physiological cost, resulting in a reduced number of secondary cases generated. However, these costs may be mitigated by secondary, so-called compensatory mutations. We attempted to identify biological pathways mutated more frequently than expected by chance in multidrug-resistant (MDR: resistance against rifampicin (RIF) and isoniazid (INH)) M. tuberculosis strains which generated secondary cases, compared to strains which did not transmit frequently. We identified nucleotide biosynthesis pathways as more frequently mutated than expected by chance in strains that transmit frequently, potentially indicating a connection to rifampicin-resistance and altered nucleotide requirements of RIF-resistant strains.
In 2017, 560000 cases of RIF-resistant M. tuberculosis infections were reported. The incidence of RIF-resistant TB cases is globally not equally distributed. For instance, countries of the former Soviet Union are heavily affected by drug-resistant variants for reasons not well understood. To shed more light onto the evolution of drug-resistant variants in the former Soviet Union, we analysed a nation-wide collection of clinical MDR M. tuberculosis strains, isolated between 2011 and 2013 in Georgia. We identified prisons as hotspots for transmission of MDR-TB. Furthermore, strains belonging to lineage 2 were more prone to being found in transmission clusters vs. strains from lineage 4. Finally, strains carrying fitness-enhancing compensatory mutations were found to transmit frequently.
Since the discovery that single drug regimens for the treatment of M. tuberculosis infections quickly lead to resistance, TB is treated with combination therapies. The current WHO-endorsed regimen for drug susceptible TB contains four drugs, including RIF and INH. Patient non-adherence and pharmacogenomic variation among patients have been demonstrated to generate sub-inhibitory drug concentrations, which in turn are implicated in the evolution of drug resistance. We investigate the influence of sub-inhibitory concentrations of RIF and INH alone and in combination. Out data hints at the possibility that katG mutations, conferring INH resistance, are also involved in conferring low-level RIF resistance.