Flow cytometry and real-time quantitative PCR as tools for assessing plasmid persistence.

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Plasmid maintenance without selection is challenging.
• Plasmid persistence is key to understanding gene transfer, especially for multidrug resistance plasmids.
• Current methods for monitoring plasmid persistence are cultivation-based, time-consuming, and not high-throughput.

Purpose of the Study:

• To introduce and validate flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative methods for monitoring plasmid persistence.
• To compare the performance of FCM and qPCR against the conventional plate count (PC) method.
• To assess the suitability of FCM and qPCR for high-throughput analysis of plasmid maintenance.

Main Methods:

• A model plasmid (pB10::gfp) was used to measure persistence in three Pseudomonas hosts.
• Plasmid persistence was monitored using conventional plate counts (PC), flow cytometry (FCM), and real-time quantitative PCR (qPCR).
• Performance criteria including dynamic range, resolution, and variance were compared across methods.

Main Results:

• FCM and real-time qPCR provided plasmid loss rate estimates comparable to the conventional PC method.
• Both FCM and qPCR demonstrated the ability to resolve differences in plasmid loss rates.
• The study identified advantages and disadvantages for each technique.

Conclusions:

• Flow cytometry (FCM) and real-time quantitative PCR (qPCR) are suitable alternatives to cultivation-based methods for routine plasmid persistence measurement.
• These alternative methods facilitate high-throughput analyses, advancing research on plasmid maintenance and antibiotic resistance.
• The findings support the adoption of FCM and qPCR for broader application in microbial genetics and epidemiology.