Direct and convenient measurement of plasmid stability in lab and clinical isolates of E. coli

Siyi Chen,Swaine L. Chen,Mårten Larsson,Robert C. Robinson

doi:10.1038/s41598-017-05219-x

Abstract

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.

Highlights

Plasmids are extrachromosomal genetic elements carried by many bacteria[1,2]
Other processes besides plasmid loss, such as mutation or deletion of the negative selection cassette, can enable bacteria to grow under restrictive conditions, but these may be distinguished by testing for chloramphenicol resistance or mCherry fluorescence
In two independent experiments, each with three replicate cultures, we found that there was no significant difference in the quantification of plasmid loss frequency by patching or by the naïve test for bacteria carrying pSLC-298 after 5 days (16.8–38.5% for the naïve test; 18.2–74.2% by patching 200–500 colonies per culture; p = 0.3125, Wilcoxon signed-rank test; Fig. 3c)

Summary

Introduction

Plasmids are extrachromosomal genetic elements carried by many bacteria[1,2]. Basic insights into plasmid biology have been transformative for molecular biology and biotechnology. This synthetic plasmid (Fig. 1a), carrying the negative selection cassette in addition to several features convenient for validating its presence and loss (including a positively selectable antibiotic marker conferring chloramphenicol resistance; two inducible fluorescent markers (mCherry under the control of the Lac promoter; GFP under the control of the arabinose promoter); and unique restriction sites for insertion of additional modules), was synthesized with the replication origin and segregation system from the E. coli/Salmonella R1 plasmid[25].

Results

Conclusion