Genomics approaches to elucidating pathways to antibiotic resistance in Neisseria gonorrhoeae

PROJECT SUMMARY/ABSTRACT The rise of antibiotic resistant bacteria poses a grave threat to public health. To outcompete susceptible bacteria and increase in prevalence, resistant strains must mitigate fitness costs incurred by resistance- conferring mutations and genes. However, not every strain of a bacterial species can acquire and maintain genetic determinants of resistance equally well, yielding a complex evolutionary landscape between susceptibility and resistance. Elucidating the nature and diversity of the mechanisms that support acquisition and maintenance of resistance will allow us to understand how resistant strains emerge and spread and thereby accelerate development of desperately needed new strategies to prevent and treat resistant infections. We focus on the clinically important pathogen Neisseria gonorrhoeae (the gonococcus), given its high burden of disease (nearly 700,000 reported cases in the US in 2021), the imminent threat of untreatable infection, and the ease of experimental manipulation. Our goal is to define the genetic networks that support acquisition and maintenance of resistance to the most clinically important antibiotics for N. gonorrhoeae treatment: the last approved antibiotic, ceftriaxone; ciprofloxacin, for which rapid diagnostics for directed therapy are approved in Europe; and the two drugs in Phase 3 trials, zoliflodacin and gepotidacin. To do so, we will leverage a dataset that includes genome sequences and antibiotic susceptibility profiles for ~18,000 clinical isolates and a library of hundreds of selected clinical isolates, representing the species diversity. We will use computational and experimental methods, including population genetics and statistical tools to identify genetic differences in sub- populations; high-throughput bar-coded library and experimental evolution to define the loci that impact resistance acquisition as a function of genetic background; genome manipulation to validate links between genotype and resistance phenotype; and mechanistic studies to elucidate how these genetic loci influence the fitness landscape between susceptibility and resistance. We expect that the results from these studies will define the stepping-stone mutations that contribute to acquisition and maintenance of resistance to the antibiotics of highest clinical importance in N. gonorrhoeae. These results can be applied to improving clinical management strategies and public health surveillance efforts. Moreover, the system we establish here can be used to further probe the biology of N. gonorrhoeae and provides a framework for the development of similar systems to dissect the genetic networks of resistance in other bacterial pathogens.