Discrimination between live and dead cellsin bacterial communities from environmental water samples analyzed by 454...

Area of Science:

• Environmental microbiology
• Molecular ecology
• Next-generation sequencing

Background:

• Propidium monoazide (PMA) is increasingly used to differentiate viable from non-viable microbial cells by targeting intact cell membranes before PCR amplification.
• Understanding the influence of environmental stressors, such as elevated temperatures, on microbial community composition is crucial for ecological assessments.

Purpose of the Study:

• To evaluate the impact of PMA treatment on 454 pyrosequencing profiles of environmental water samples subjected to varying heat stress.
• To assess the combined effect of PMA and heat treatment on microbial community structure in canal water and seawater.

Main Methods:

• Genomic DNA extraction from environmental water samples (canal and seawater).
• Propidium monoazide (PMA) treatment of samples exposed to different temperatures (ambient, 50°C, 60°C, 85°C).
• 16S rRNA gene amplification using universal bacterial primers and 454 pyrosequencing.
• Microbial community analysis and comparison of sequence profiles.
• BacLight LIVE/DEAD staining and flow cytometry to confirm membrane integrity changes.

Main Results:

• PMA treatment showed minimal impact on non-heated samples, preserving the original microbial community profiles.
• Elevated temperatures significantly altered the relative abundance of bacterial groups, with a more pronounced effect in heated seawater compared to heated canal water.
• High-temperature exposure (85°C) combined with PMA treatment completely inhibited PCR amplification, indicating severe membrane damage.
• Flow cytometry confirmed increased propidium iodide uptake in heat-stressed samples, validating the effect on cell membrane integrity.

Conclusions:

• PMA treatment is effective in distinguishing between viable and non-viable cells, but its application requires careful consideration of environmental conditions.
• Heat stress significantly impacts microbial community structure in aquatic environments, affecting membrane integrity and subsequent molecular profiling.
• The study highlights the importance of accounting for temperature-induced changes in cell viability when using PMA-qPCR or sequencing for microbial community analysis.