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Measuring Drug-Induced Changes in Metabolite Populations of Live Bacteria: Real Time Analysis by Raman Spectroscopy
Paul R Carey1, Grant R Whitmer1, Michael J Yoon1
1Department of Biochemistry , Case Western Reserve University School of Medicine , 10900 Euclid Avenue , Cleveland Ohio 44106 , United States.
The Journal of Physical Chemistry. B
|May 25, 2018
Summary
Raman spectroscopy reveals real-time molecular changes in E. coli treated with antibiotics or hydrogen peroxide. Key findings include depletion of proteins and nucleic acids, and altered metabolite levels, such as increased citrate.
Area of Science:
- Biochemistry
- Microbiology
- Spectroscopy
Background:
- Bacterial responses to antibiotics and oxidative stress involve complex molecular changes.
- Understanding these changes in real-time is crucial for developing new treatments and diagnostics.
Purpose of the Study:
- To investigate the molecular alterations in *Escherichia coli* (*E. coli*) cells upon exposure to various antibiotics and hydrogen peroxide using Raman difference spectroscopy.
- To identify specific molecular species and metabolic pathways affected by these treatments.
Main Methods:
- Utilizing Raman difference spectroscopy to analyze *E. coli* cells treated with chloramphenicol, dihydrofolate reductase inhibitors, meropenem, or hydrogen peroxide.
- Employing a sample preparation method involving drug infusion, washing, freezing, and lyophilization of cells before Raman microscopy.
- Analyzing time-dependent difference spectra ([treated cells] - [untreated cells]) to track intracellular species kinetics.
Main Results:
- Raman difference spectroscopy provided detailed molecular insights into bacterial cell changes.
- Observed real-time depletion of protein and nucleic acid populations.
- Detected reproducible Raman features attributed to altered cell metabolite populations, with a notable increase in citrate levels.
- Confirmed citrate's role by observing a lack of the corresponding Raman feature in a *gltA* mutant strain.
- Noted a strong resemblance in Raman difference spectra between meropenem and hydrogen peroxide treated cells.
Conclusions:
- Raman difference spectroscopy is a powerful tool for real-time molecular analysis of bacterial responses to antimicrobial agents and oxidative stress.
- Antibiotic and hydrogen peroxide treatments induce significant changes in bacterial protein, nucleic acid, and metabolite levels.
- Citrate accumulation is a potential metabolic response to certain cellular stresses in *E. coli*.
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