Evolutionary dynamics of antibiotic persistence and resistance: Novel insights from high-throughput experimental evolution

A rapidly growing number of patients succumb to bacterial infections for which effective treatment is lacking. The major culprit of this lurking worldwide health crisis is the growing ability of bacteria to cope with antibiotic stress. Due to their high reproduction rates and vast population sizes, bacteria exhibit an enormous potential for evolutionary adaptation. As a consequence, antibiotic survival strategies emerge and spread at staggering rates, circumventing many human attempts to subdue infectious diseases. Although the therapeutic challenges posed by bacterial evolution are widely recognized, knowledge on the evolutionary dynamics of antibiotic survival strategies remains fragmentary. A better understanding of evolutionary adaptation to antibiotic stress is required for an accurate prediction and successful prevention of therapy failure. This doctoral study presents novel insights into the evolutionary dynamics of antibiotic resistance and persistence, two major strategies adopted by bacteria to prevent drug-induced lethality. Resistance involves genetic alterations that allow bacteria to maintain active growth in the presence of antibiotics, and has been reported towards virtually all antibiotics currently in use. Nonetheless, many infectious populations are not resistant but still challenging to treat due to the presence of a small number of antibiotic-tolerant persister cells. These cells are genetically susceptible, but phenotypically tolerant to antibiotic treatment. As persister cells allow a bacterial population to re-establish once treatment is ceased, they are considered a major cause of recurrent and chronic infections. Both resistance and persistence render a bacterial population refractory to antibiotic treatment, yet important differences between both strategies result in distinct patterns of emergence and spread. Using high-throughput experimental evolution, we identified several factors that influence the evolutionary dynamics of resistance and persistence, including population bottlenecks and concentrations of nutrients and antibiotics experienced by bacterial populations. Population bottlenecks, which are frequently encountered by infecting pathogens during within-host or between-host transmissions, are shown to slow down evolutionary adaptation, reduce genetic diversity, and promote population divergence. Furthermore, we demonstrate that antibiotic treatment applied under nutrient-rich conditions favors resistance over persistence, while persistence emerges more readily when the antibiotic concentration is high. Although these findings still await translation to clinical settings, they point out major determinants of evolution that need to be considered in the development of novel therapeutic strategies. Despite the increasingly recognized relevance of both resistance and persistence, evolutionary interactions between these strategies are poorly understood. Through large-scale phenotyping of evolved populations, combined with theoretical simulations, we demonstrate that resistance and persistence are heavily intertwined in an evolutionary context. In environments where both strategies provide a selective advantage, resistance and persistence can evolve simultaneously. We hypothesize that, while resistance is most beneficial in this case, persistence mutations can arise more easily due to a larger mutational target and/or effect size, and can act as a stepping stone towards high-level resistance. This hypothesis was further investigated by selecting for resistance in strains with a range of initial persistence levels. High persistence levels were found to accelerate resistance evolution through an increased viable cell pool and an increased mutation rate. A novel mathematical model predicts that this persistence-resistance link has clinical implications for the clearance of bacterial infections. The development of innovative therapies that prevent resistance and persistence evolution would benefit from a deepened insight into the underlying molecular mechanisms. Compounds that stimulate persister awakening hold promise for the eradication of persister cells, and could ultimately slow down resistance development. However, studying persister awakening requires single-cell approaches, which are challenging due to the transient and rare nature of persister cells. We present an optimized experimental procedure to enrich persisters, enabling single-cell studies of persister awakening at a much higher throughput than previously reported. In conclusion, the presented results provide novel insights in bacterial adaptation to antibiotic stress, and uncover important interactions between resistance and persistence. These findings do not only emphasize the need for anti-persister therapies, but might also contribute to the development of novel therapeutic strategies that impede evolutionary adaptation to antibiotic treatment.

Jaar van publicatie:2021

Toegankelijkheid:Closed