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Updated: Dec 31, 2025
Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli
Published on: March 24, 2023
The exclusive use of flow cytometry to evaluate the antibiotic-susceptibility
Takashi Soejima1, Jun-ichi Minami, Keiji Iwatsuki
1Department of Bacteriology, Kyushu University, Higashi-ku, Fukuoka, Japan. t_soezim@morinagamilk.co.jp
Background:
Live and injured bacteria cannot be successfully discriminated using flow cytometric methods (FCM) with commercial live/dead staining agents because injured cells have intact cell membranes and are counted as live cells. We previously reported that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro (Microbiol. Immunol. 51:763-775, 2007).
Methods:
We report that EMA cleaves the chromosomal DNA of antibiotic-injured, but not live, Listeria monocytogenes. The combination of FCM and EMA treatment was evaluated as a rapid method to discriminate between live and antibiotic-injured L. monocytogenes. Additionally, we evaluated our methodology using blood from pediatric patients infected with other gram-negative and gram-positive bacteria.
Results:
For antibiotic-injured, but not live, L. monocytogenes in blood, photoactivated EMA suppressed SYTO9 staining, as the SYTO9 staining of the antibiotic-injured L. monocytogenes was weak compared with that of live cells. Similarly, the rapid and clear discrimination between live and injured bacteria (gram-negative and gram-positive) was performed using the blood of pediatric patients administered antibiotics.
Conclusions:
The combination of FCM with EMA treatment is a rapid method for evaluating the susceptibility of live pathogens in infants with bacteremia without the need for bacterial culture.
General Significance:
This assay is more rapid than other currently available techniques due to the elimination of the time-consuming culture step and could be used in clinical settings to rapidly determine the success of antibiotic treatment in pediatric bacteremia through the discrimination of injured (i.e., susceptible to the administered antibiotics) and live pathogens.
Insights
This study introduces a rapid method using photoactivated ethidium monoazide (EMA) with flow cytometry to distinguish live from antibiotic-injured bacteria, crucial for treating infant bacteremia.
Area of Science:
- Microbiology
- Clinical Diagnostics
- Flow Cytometry
Background:
- Current flow cytometry methods struggle to differentiate live from injured bacteria due to intact cell membranes in injured cells.
- Previously, photoactivated ethidium monoazide (EMA) was shown to directly cleave bacterial DNA.
Purpose of the Study:
- To evaluate the combination of flow cytometry and EMA treatment as a rapid method for discriminating between live and antibiotic-injured bacteria.
- To assess the methodology in blood samples from pediatric patients with bacterial infections.
Main Methods:
- Utilizing photoactivated EMA to target and cleave DNA in antibiotic-injured bacteria.
- Employing flow cytometry (FCM) to analyze bacterial populations after EMA treatment.
- Testing the method on Listeria monocytogenes and diverse bacteria from pediatric blood samples.
Main Results:
- Photoactivated EMA selectively cleaved chromosomal DNA in antibiotic-injured Listeria monocytogenes, but not in live cells.
- EMA treatment suppressed SYTO9 staining in injured bacteria, enabling clear discrimination from live bacteria via FCM.
- The method successfully differentiated live from injured gram-negative and gram-positive bacteria in pediatric blood samples.
Conclusions:
- The FCM and EMA combination offers a rapid diagnostic tool for assessing antibiotic susceptibility in infant bacteremia without culturing bacteria.
- This technique significantly reduces assay time compared to traditional methods, enabling faster clinical decisions on antibiotic treatment efficacy.
