Genomic Insight into Primary Adaptation of Mycobacterium tuberculosis to Aroylhydrazones and Nitrofuroylamides In Vitro

<b>Background/Objectives:</b> New anti-tuberculosis compounds are needed to treat patients infected with multi- or extensively drug-resistant <i>Mycobacterium tuberculosis</i> strains. Studies based on spontaneous in vitro mutagenesis can provide insights into the possible modes of action and resistance mechanisms of such new compounds. We evaluated the primary response of <i>M. tuberculosis</i> in vitro to the action of new aroylhydrazones and nitrofuroylamides. <b>Methods</b>: The reference strain H37Rv was cultured on solid media with compounds at increased concentrations relative to MIC. Resistant clones were investigated using whole-genome sequencing and bioinformatics tools to assess the role and potential impact of identified mutations. <b>Results</b>: Some of the mutations are significant (based on in silico analysis), located in essential genes, and therefore of particular interest. Frameshift mutations were observed in (i) <i>Rv2702/ppgK</i>, which is associated with starvation-induced drug tolerance and persistence in mice, and (ii) <i>Rv3696c/glpK</i>, which has been described as a switch on/off mutation associated with drug tolerance. Nonsynonymous substitutions were found in <i>Rv0506/mmpS2</i>, which belongs to the Mmp protein family involved in transport and drug efflux, and in <i>infB</i>, encoding the translation initiation factor IF-2. <b>Conclusions</b>: The primary adaptation of <i>M. tuberculosis</i> to the selective pressure of the tested compounds is complex and multifaceted. It involves multiple unrelated genes and pathways linked to non-specific drug tolerance, efflux systems, or mechanisms counteracting oxidative stress.