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Published on: July 17, 2013
Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous
Patricia J Hare1, Jonathan I Batchelder2, Travis J LaGree2
1Department of Molecular Biology & Biophysics, UConn Health; School of Dental Medicine, UConn Health.
Abstract:
Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to underlie clinical treatment failure. Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited. This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus) during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.
Insights
This study presents a new time-lapse imaging method to track antibiotic persister cells in Pseudomonas aeruginosa and Staphylococcus aureus. This approach allows detailed observation of individual bacterial cell survival and resuscitation during antibiotic treatment.
Area of Science:
- Microbiology
- Cell Biology
- Antimicrobial Resistance
Background:
- Antibiotic persistence allows a small fraction of susceptible bacteria to survive antibiotic treatment, contributing to treatment failure.
- Single-cell studies using time-lapse fluorescence microscopy have advanced understanding of persister cells in Escherichia coli.
- Studying persister cells in other key pathogens using similar high-resolution methods has been limited.
Purpose of the Study:
- To develop and validate an adaptable time-lapse imaging approach for studying antibiotic persister cells in Pseudomonas aeruginosa and Staphylococcus aureus.
- To enable detailed observation of individual bacterial cell responses to antibiotics at the single-cell level.
- To provide a resource for investigating persister cell formation, survival, and resuscitation mechanisms in clinically relevant pathogens.
Main Methods:
- Adaptation of time-lapse fluorescence microscopy techniques for Pseudomonas aeruginosa and Staphylococcus aureus.
- Introduction of fluorescent reporters (transcriptional and translational) and dyes to label cellular features.
- Observation and tracking of individual bacterial cell phenotypes, morphology, and gene expression during and after antibiotic treatment.
Main Results:
- Successful implementation of time-lapse imaging for observing antibiotic persister cells in both Gram-negative (P. aeruginosa) and Gram-positive (S. aureus) bacteria.
- Detailed tracking of individual persister cell resuscitation and phenotypic changes post-antibiotic exposure.
- Demonstration of the utility of single-cell imaging for understanding persister cell dynamics beyond population-level assays.
Conclusions:
- The developed adaptable imaging approach facilitates high-resolution studies of antibiotic persister cells in key clinical pathogens.
- This method provides crucial insights into the survival and recovery mechanisms of individual persister cells.
- This work serves as a valuable resource for advancing research on antibiotic resistance and treatment strategies.
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