Investigating antibiotic susceptibility and persister formation in Yersinia pseudotuberculosis

Abstract

Antibiotic resistance is mediated by virulence mechanisms. While genetic antibiotic resistance can be detected by standard susceptibility assays, subpopulations of phenotypically resistant cells, such as antibiotic persisters, evade detection. Phenotypic resistance could hinder first-line antibiotic efficacy, an increasing concern due to the threat of bioterrorism, where antibiotic resistant pathogens could be exploited in deliberate outbreaks. Yersinia pestis is a pathogen listed by the CDC as a Category A biological agent. Exploitation of resistant strains substantially amplifies the devastation caused by a deliberate attack and complicates treatment efforts. Y. pestis is thought to have evolved from the less virulent Yersinia pseudotuberculosis up to 7000 years ago. Due to their high genetic similarity, Y. pseudotuberculosis can be used as a safe model organism to investigate Y. pestis, including its antibiotic resistance mechanisms. The aim of this project was to investigate the susceptibility of Y. pseudotuberculosis to clinically relevant antibiotics, and its ability to form antibiotic persisters. I found that Y. pseudotuberculosis was most susceptible to ceftriaxone and least susceptible to gentamicin and chloramphenicol. I further demonstrated that Y. pseudotuberculosis persisters were found after exposure to levofloxacin, ciprofloxacin and ceftriaxone. Finally, I assessed whether Y. pseudotuberculosis persisters were tolerating ceftriaxone and levofloxacin through antibiotic efflux and, if so, whether the efflux pump inhibitors sertraline and verapamil were capable of inhibiting efflux and reducing the persister survival fraction. While the addition of verapamil did not increase the killing of persister cells, the addition of sertraline to ceftriaxone significantly increased the killing of persister cells compared to ceftriaxone alone. Taken together, my results provide valuable evidence and better characterisation of the antibiotic susceptibility of Y. pseudotuberculosis, its persister-forming capabilities and its ability to efflux antibiotics.