Flow Cytometry and Real-Time Quantitative PCR as Tools for Assessing Plasmid Persistence

Abstract

The maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is timeconsuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in three Pseudomonas hosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses.

Synthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio cholerae

“…Since only cells will be stained and not small particles this allows correct quantification of GFP ‐ cells. The second challenge described in the published study is the overestimation of replicon stability caused by continued fluorescence of the stable GFP after replicon loss [65]. The obvious solution to this problem we present here is the use of unstable GFP variants.…”

“…While GFP encoded on secondary replicons was used before to detect respective copy numbers we use it here to measure replicon stability [36]. A very recent study describes a similar approach [65]. One challenge they experienced is the separation of GFP ‐ cells from background caused by small particles in the used buffer.…”

In Vivo and In Vitro Detection of Luminescent and Fluorescent Lactobacillus reuteri and Application of Red Fluorescent mCherry for Assessing Plasmid Persistence

“…As an example, the stability data for 6475-CBRluc-mCherry generated by both FCM and PC were similar and indicated 100% plasmid loss at day 4 ( Fig 3A ). Loftie-Eaton et al have also found that the plasmid persistence profiles generated by these techniques are similar [ 34 ]. However, the plasmid loss rate of 6475- mCherry detected by PC was clearly higher than the rate shown by FCM.…”

Persistence of antibiotic resistance plasmids in bacterial biofilms

“…The fraction of plasmid‐bearing cells in each culture was estimated via quantitative PCR (qPCR; Loftie‐Eaton, Tucker, Norton, & Top, ) of the plasmid‐encoded trfA gene (encoding the replication initiation protein) and the chromosomally encoded 16S rRNA genes. Plasmid pB10 has a low copy number (~2 per cell, data not shown), and the number of 16S rRNA gene copies in Acinetobacter baumannii is five (Maslunka, Carr, Gürtler, Kämpfer, & Seviour, ; Stoddard, Smith, Hein, Roller, & Schmidt, ).…”