Plasmids are important mobile elements in bacteria, contributing to evolution, virulence, and
antibiotic resistance. Natural plasmids are generally large and maintained at low copy number and
thus prone to be lost. Therefore, dedicated plasmid maintenance systems have evolved, leading to
plasmid loss rates as low as 1 per 107 divisions. These low rates complicate studies of plasmid loss, as
traditional techniques for measuring plasmid loss are laborious and not quantitative. To overcome these
limitations, we leveraged a stringent negative selection system to develop a method for performing
direct, quantitative measurements of plasmid loss in E. coli. We applied our method to gain mechanistic
insights int... More
Plasmids are important mobile elements in bacteria, contributing to evolution, virulence, and
antibiotic resistance. Natural plasmids are generally large and maintained at low copy number and
thus prone to be lost. Therefore, dedicated plasmid maintenance systems have evolved, leading to
plasmid loss rates as low as 1 per 107 divisions. These low rates complicate studies of plasmid loss, as
traditional techniques for measuring plasmid loss are laborious and not quantitative. To overcome these
limitations, we leveraged a stringent negative selection system to develop a method for performing
direct, quantitative measurements of plasmid loss in E. coli. We applied our method to gain mechanistic
insights into a heterologously reconstituted segregation system in lab strains and clinical isolates
of E. coli. We also performed direct stability studies of a currently circulating resistance plasmid in a
clinical isolate, strain EC958, which is a member of the rapidly expanding global ST131 E. coli clone.
Our results establish the foundational assays required to screen for small molecules targeting plasmid
stability, which could complement current strategies for reducing the spread of antibiotic resistance,
complementing other strategies for treating antibiotic resistant bacteria.
