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Published on: May 5, 2017
Rapid Antibiotic Susceptibility Determination for Yersinia pestis Using Flow Cytometry Spectral Intensity Ratio (SIR)
Eran Zahavy1, Shahar Rotem2, David Gur2
1Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel. eranz@iibr.gov.il.
Abstract:
Rapid antimicrobial susceptibility tests (ASTs) are essential tool for proper treatment of patients infected by Yersinia pestis (Y. pestis), the causative agent of plague, or for post-exposure prophylaxis of a population exposed to a naturally acquired or deliberately prepared resistant variant. The standard AST of Y. pestis is based on bacterial growth and requires 24-48 h of incubation in addition to the time required for prior isolation of a bacterial culture from the clinical or environmental sample, which may take an additional 24-48 h. In this study, we present a new and rapid AST method based on a fluorescence determination of the minimum inhibitory concentration (MIC). Our method includes the incubation of bacteria with an antibiotic, followed by staining of the bacteria with oxonol dye (SynaptoGreen C4/FM1-43), which enables the rapid detection of an antibiotic's effect on bacterial viability. We show that stained, non-viable bacteria exhibit a spectral redshift and an increase in fluorescence intensity compared to intact control bacteria. Based on these criteria, we developed a rapid flow cytometer measurement procedure and a unique spectral intensity ratio (SIR) analysis that enables determination of antibiotic susceptibility for Y. pestis within 6 h instead of the 24 to 48 h required for the standard AST. This new rapid determination of antibiotic susceptibility could be crucial for reducing mortality and preventing the spread of disease.
Insights
A new rapid antimicrobial susceptibility test (AST) for Yersinia pestis significantly cuts detection time from 48 hours to 6 hours. This faster method aids in timely plague treatment and public health response to antibiotic resistance.
Area of Science:
- Microbiology
- Infectious Diseases
- Biotechnology
Background:
- Yersinia pestis (Y. pestis) causes plague, necessitating rapid antimicrobial susceptibility tests (ASTs) for effective treatment.
- Standard Y. pestis ASTs rely on bacterial growth, requiring 24-48 hours post-isolation, delaying critical therapeutic decisions.
Purpose of the Study:
- To develop and validate a novel, rapid AST method for Y. pestis.
- To significantly reduce the time required for determining antibiotic susceptibility in Y. pestis.
Main Methods:
- Incubation of Y. pestis with antibiotics, followed by staining with oxonol dye (SynaptoGreen C4/FM1-43).
- Detection of changes in bacterial viability using fluorescence intensity and spectral redshift.
- Development of a flow cytometer procedure and spectral intensity ratio (SIR) analysis for rapid MIC determination.
Main Results:
- Non-viable Y. pestis bacteria exhibit increased fluorescence intensity and spectral redshift upon staining.
- The new method accurately determines Y. pestis antibiotic susceptibility within 6 hours.
- This represents a substantial time reduction compared to standard 24-48 hour AST methods.
Conclusions:
- The rapid fluorescence-based AST offers a crucial advancement for managing Y. pestis infections.
- Faster susceptibility results can improve patient outcomes and control disease spread.
- This method is vital for addressing both natural and engineered antibiotic resistance in Y. pestis.
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