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  current news   Press   selected story    
     
  13 July 2017  
 
Direct and convenient measurement of plasmid stability in lab and clinical isolates of E. coli
 
 




Authors
Siyi Chen1, Mårten Larsson2,6, Robert C. Robinson2,4,5 & Swaine L. Chen1,3

1  Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, 1E Kent    Ridge Road, NUHS Tower Block, Level 10, Singapore, 119228, Singapore.
2  Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), 61    Biopolis Drive, Singapore, 138673, Singapore.
3  GERMS and Infectious Diseases Group, Genome Institute of Singapore, 60 Biopolis Street, Genome,    #02-01, Singapore, 138672, Singapore.
4  Department of Biochemistry, National University of Singapore, Singapore, 117597, Singapore. 5NTU    Institute of Structural Biology, Nanyang Technological University,
5  9 Nanyang Drive, Singapore, 636921, Singapore.
6  Present address: Department of Medical Biochemistry and Microbiology, Uppsala University, SE-751 23,    Uppsala, Sweden.

Published online in Scientific Reports on 6 July 2017.
https://www.nature.com/articles/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.

Figure

Figure legend

Direct measurement of pEC958 plasmid stability in its native host strain, EC958. (a) Genetic map of plasmid pEC958 indicating the location of genes (highlighted in gray) replaced by the negative selection cassette. (b) Plasmid stability measured in EC958. Raw plasmid loss frequencies (assayed using the single colony test, white bars) or rates (assayed using the Luria-Delbrück test, black bars) are plotted with error bars as in Fig. 3. Numbers above each bar indicate the percentage of colonies growing that are truly plasmid-free based on antibiotic resistance and PCR for plasmid-specific sequences. Significant differences in plasmid loss frequency were calculated relative to pSLC-344 (pEC958 ∆a0138) using a 2-tailed Student’s t-test on log transformed values. ns indicates p > =0.05; *p < 0.05; ***p < 0.001. Plasmid loss frequency data are plotted as the mean of log-transformed values from at least three independent experiments with error bars indicating standard deviations, while the Luria-Delbrück rate data are plotted as the mean of log-transformed values from at least two independent experiments with error bars depicting the range between the two values. Significance was tested for differences in Luria-Delbrück rates only when > = 3 experimental replicates were performed.

For more information on Robert ROBINSON's lab, please click here.