Sandra Marina Wellner: Identification of antibiotic fitness-genes in aminoglycoside-resistant Escherichia coli
Email: sandra.wellner@sund.ku.dk
https://innotargets.ku.dk/fellows/sandra-wellner/
THE PROJECT
The discovery of antibiotics has saved millions of lives and revolutionized modern medicine. However, the extensive use of antibiotics in human and veterinary medicine has led to an increased spread of antibiotic resistance among pathogens. Multi-drug resistant Gram-negative bacteria such as Enterobacteriaceae including Escherichia coli are of major concern to global health, especially since the antibiotic discovery pipeline for Gram-negative pathogens is drying out. Therefore, innovative approaches employing advanced technologies are crucial. One possibility among them is the discovery and utilization of antimicobial adjuvants, which are drugs that potentiate the already existing antimicrobials.
THE PURPOSE
The purpose of this PhD thesis was to gain an insight into the cellular adaptations that take place in aminoglycoside-resistant E. coli during aminoglycoside stress. This understanding could be the key to the identification of novel aminoglycoside helper- compound targets.
THE RESULTS
In this project, 106 aminoglycoside fitness-genes were identified, which were important for growth during streptomycin, gentamicin and neomycin stress in aminoglycoside- resistant E. coli strains using a TraDIS-based approach for screening out mutants. These fitness-genes emphasized the role of the membrane, bacterial stress response, ATP metabolism, cell division and Enterobacterial Common Antigen biosynthesis under aminoglycoside pressure and represent potential candidates for helper-drug design. Furthermore, a novel sequencing-based competition approach was employed in this work to verify the TraDIS results, which allowed the direct comparison of the growth between wild type and mutant strains.
Furthermore, this work uncovered that deletion of pcnB led to an increase in antibiotic susceptibility towards aminoglycosides, ampicillin, and tetracycline in E. coli harboring ColE1-family resistance plasmids. This observation was attributed to the involvement of PcnB, an enzyme which polyadenylates RNA, in the antisense RNA mechanism used by ColE1-type plasmid to regulate their plasmid copy number. Hence, PcnB could be targeted to reduce the copy number of ColE1- type plasmids and was suggested as helper-drug target to re-sensitize resistant bacteria harboring ColE1-family plasmids to antibiotics.
Additionally, two antibiotics, which potentiate the effect of neomycin in aminoglycoside-resistant E. coli, rifamycin O and thermorubin, were identified in an aminoglycoside helper-drug screen. Moreover, in a screen using 880 extracts of Actinomycetes, one extract with moderate general antimicrobial properties and one with moderate gentamicin helper-compound activity were identified, showing the diverse usability of the described helper-compound screening approach.