Mycobacterium tuberculosis resistance evolution

Abstract

With nearly half a million new multidrug resistant (MDR) tuberculosis (TB) cases estimated annually, TB is a major contributor to the current antimicrobial resistance crisis. MDR-TB is defined as an infection with a Mycobacterium tuberculosis complex (MTBC) strain resistant to the two most effective first-line drugs rifampicin (RIF) and isoniazid. To effectively address this public health crisis, it is essential to un-derstand resistance mechanisms and evolution. This will better guide the development of new antibiot-ics and treatment strategies, and avoid rapid resistance development, as seen with bedaquiline (BDQ). This work explores resistance mechanisms to one key MDR-TB treatment drug, namely BDQ, and new evolutionary medicine informed treatment strategies based on negative hysteresis. BDQ, one of the most important drugs for treating MDR-TB, is facing increasing resistance in clinical MTBC strains in several high incidence settings. Clinical MTBC strains acquire BDQ resistance through mutations in the atpE gene, which encodes the ATP synthase (primary target of BDQ), and in the Rv0678 gene, which regulates the mmpS5-mmpL5 efflux pump system. Most clinical resistance is associated with mutations in Rv0678, leading to increased expression of the efflux pump genes. In this work, RNA sequencing (RNAseq) analysis of BDQ-resistant mutants, harbouring different mutations in Rv0678 (to-tal loss-of-function mutations and reduced-function mutations), was conducted. The results revealed that efflux pump expression varied among clones, with baseline mmpS5-mmpL5 efflux pump gene ex-pression upregulated without BDQ exposure. Surprisingly, under BDQ challenge, the wild-type H37Rv strain upregulated the mmpS5-mmpL5 efflux pump to similar levels as the Rv0678 mutant clones, point-ing to other mechanisms involved in resistance development. Analysis of the transcriptional response of Rv0678 mutant clones and H37Rv at different BDQ exposure timepoints revealed a complex response indicating that resistance is mediated not by a single mechanism, but rather by an enhanced metabolic plasticity of the Rv0678 mutants, which allows them to shift to a dormant-like state under stress while maintaining some cellular functions. The second focus of this work was to explore evolution-informed treatment strategies aimed at en-hancing drug efficacy and circumvent resistance evolution by using negative hysteresis as potential mechanism in sequential treatments to limit the evolutionary potential of MTBC strains. An in vitro model was designed based on short pre-exposure with one drug, followed by main-exposure with the second drug utilizing sub-lethal concentrations which potentially induces cellular hysteresis, a phenom-enon where antibiotic effectiveness is enhanced based on the order of drug administration. Sequential exposure of MTBC reference strain H37Rv with ethambutol (EMB) followed by RIF led to a sequence-dependent reduction in bacterial load, attributed to the negative hysteresis effect, where EMB pre-exposure enhanced RIF's efficacy. RNAseq was employed to investigate the underlying mechanisms, indicating that the cellular hysteresis effect, induced by the respective drug sequence, was likely driven by a cell surface stress response and metabolic remodelling by a danK-mediated stress response. Taken together, the results obtained in this thesis contradicts common knowledge that BDQ resistance induced by Rv0678 mutations is solely conferred by upregulation of the mmpS5-mmpL5 efflux pump system only, but rather points to the role of an enhanced metabolic plasticity in antibiotic responses as important survival strategy. It also shows, that comprehensive studies based on mutant generation combined with OMICS tools are needed to understand pathogen drug response, and escape mecha-nisms. Further, to the best of our knowledge, for the first time the potential of negative hysteresis in-duced by fast drug switching for enhancing drug efficacy even at sub-minimum inhibitory concentration (MIC) conditions was observed in M. tuberculosis. This has great potential for optimizing TB treatment concepts especially for drug resistant MTBC strains, where drug options are limited and cycling thera-pies with a lower number of antibiotics significantly enhance treatment possibilities. Accordingly, evolu-tionary informed treatment strategies should be urgently further exploited.