Demonstrating plasmid-based horizontal gene transfer in complex environmental matrices: a practical approach for a...

Area of Science:

• Environmental microbiology
• Molecular biology
• Public health

Background:

• Plasmid-based spread of antibiotic resistance genes (ARGs) in environmental microbial communities poses a public health risk.
• Studying plasmid transfer in natural environments is methodologically challenging.

Purpose of the Study:

• To compare three common methods for evaluating plasmid transfer in natural microbial communities.
• To identify the limitations of culture-based, fluorescence-based, and molecular-based approaches for detecting plasmid dissemination.

Main Methods:

• Utilized the broad host range plasmid pB10 for comparative analysis.
• Evaluated culture-based selection, fluorescence-based detection, and molecular-based approaches.
• Assessed plasmid transfer under varying initial donor inocula and environmental conditions.

Main Results:

• Culture-based methods were hindered by pre-existing antibiotic resistance in natural communities, requiring high donor inocula.
• Fluorescence-based detection was limited by the narrow host range of modified plasmids and high background fluorescence.
• Molecular-based approaches proved effective for detecting rare plasmid transfer events with low, realistic donor inocula.

Conclusions:

• The molecular-based approach is the most suitable for studying rare plasmid transfer events in environmental settings.
• Successful plasmid transfer detection, regardless of method, depends on donor inoculum stability.
• Eukaryotic predation significantly influences plasmid transfer dynamics in different environmental matrices.