Onderzoek naar overlevingsmechanismen tegen geneesmiddelen in Mycobacterium abscessus

Bacterial infections remain a formidable global health challenge, placing substantial burden on healthcare systems and economies. The emergence of strains capable of circumventing antibiotic treatments exacerbates this threat. While tuberculosis persists as a major concern in developing countries, Mycobacterium abscessus is increasingly recognized for its recalcitrance and severe clinical outcomes, particularly in immunocompromised individuals. Despite its growing clinical significance, the molecular basis underlying the exceptional resilience of M. abscessus remains poorly understood, underscoring the need for further mechanistic insights that can inform the development of more effective therapeutic strategies. This dissertation addresses these gaps by employing an experimental evolution approach to examine how M. abscessus adapts and develops resistance under antibiotic pressure. As this methodology was previously uncharted for M. abscessus, initial efforts focused on establishing robust experimental conditions, such as optimizing inoculum size, growth phase, media selection, and antibiotic dosing, to balance adequate selective pressure with sufficient bacterial survival. Under high-dose amikacin-rifabutin combination exposure, rapid selection of mutations conferring high-level resistance occurred by the third to fourth treatment cycle, resulting in dual resistance to both antibiotics. Although these dominant mutations initially obscured the presence of secondary adaptive pathways, later experiments uncovered additional stress-response mutations that trigger population-wide transcriptional reprogramming, thereby promoting antibiotic survival. This work establishes a framework for using experimental evolution to explore not only high-level resistance but also stress-adaptation pathways in M. abscessus. A key finding was the identification of a single guanine deletion in the upstream regulatory region of the WhiB7 transcriptional regulator, which mediates inducible resistance to multiple antibiotic classes. This mutation leads to constitutive WhiB7 activation and confers multidrug resistance. Molecular analyses provided insights into a likely transcriptional attenuation mechanism governing WhiB7 activation in M. abscessus. Additionally, experiments with high-dose moxifloxacin selected for mutations that disrupted the σH-inhibitory protein RshA. Although these variants did not markedly affect overall drug susceptibility, they significantly enhanced bacterial survival under antibiotic stress. Subsequent barcoded gene-replacement, complementation, and knockout studies confirmed that these stress-response pathways collectively enable M. abscessus to resist treatment by inducing intrinsic resistance and promoting antibiotic tolerance. A response that further complicates treatment regimens that rely on aminoglycosides, macrolides, and fluoroquinolones. To evaluate the clinical relevance, a genomic library of 222 M. abscessus clinical isolate strains, encompassing all subspecies, was analyzed. Multiple polymorphisms were found within the WhiB7 regulatory region, although their effect on antibiotic susceptibility remains to be determined. While disruptive RshA mutations were absent in this curated set, certain σH polymorphisms correlated with increased drug sensitivity, demonstrating the importance of this pathway. Moreover, subspecies-specific synonymous variants in both the WhiB7 upstream region and the gene encoding RshA may facilitate rapid molecular typing and simultaneous profiling of relevant stress responses. In conclusion, this dissertation demonstrates that M. abscessus readily acquires multidrug resistance and antibiotic tolerance through the activation of intrinsic stress responses. Given the limited therapeutic arsenal currently available, there is an urgent need for novel drug discovery efforts. By pinpointing critical vulnerabilities in these stress responses, the findings presented here offer new avenues for the development of targeted strategies to circumvent or exploit these mechanisms, ultimately improving clinical outcomes for M. abscessus infections.