Mechanisms of adaptation to the fitness cost of antibiotic resistance

Abstract: The dissemination and persistence of antibiotic resistance, is not only depending on the volume of drugs used but also on the resistance mechanisms effect of bacterial fitness (reproductive ability). Fitness is a multifactorial parameter that is comprised of the relative growth rate of the resistant pathogen in the host and in the environment, as well as the clearance and transmission rates compared to susceptible strains. The focus of this thesis has been to determine (i) the connection between the mechanisms of resistance and their effect on growth in vitro and in a host-model, and (ii) how different adaptive mechanisms can partly or fully reverse the deleterious effects of resistance mutations. More specifically the fitness effect of mutational resistance towards the translational inhibitors mupirocin and streptomycin, targeting the isoleucyl–tRNA synthetase (IleRS) and the ribosomal 30S subunit, respectively were investigated using Salmonella typhimurium as a model organism. These studies showed that a fitness cost was associated with the resistance mutations and that suppression of their deleterious effect could be achieved by both intragenic and extragenic compensatory events. Three compensatory mechanisms that could restore fitness were identified, (i) intragenic mutations in the target protein (IleRS), (ii) extragenic (i.e. outside the target protein) mutations in the ribosomal proteins of the 30S and 50S subunits and (iii) extragenic compensatory events increasing the expression of the target protein (IleRS). The mechanism behind the resistant and compensatory mutations effect on respective target protein could be determined by in vivo and in vitro kinetical measurements of ribosomal translation (rate and accuracy) and the IleRS aminoacylation activity. The impact on activity for the resistant and compensatory mutations was shown to correlate with their effect on growth rate. However, since the fitness impact of the resistance mutations has been seen to vary between different in vitro conditions and between in vitro and in vivo conditions, we investigated and validated the nematode Caenorhabditis elegans (C. elegans) as an in vivo model for determining the fitness effects of resistance against several classes of antibiotics (including mupirocin and streptomycin). The fitness impact of the resistance mutations measured in C. elegans, correlated well with what had been detected in the mouse model of typhoid fever. It is worth noting that for all resistant strains, relative fitness in the two hostmodels was lower compared to fitness measured in the Luria Bertani broth laboratory medium. In conclusion, we have shown how resistance and compensatory mechanisms can at the protein and cellular level affect the stability of resistance in different in vitro and in vivo models.