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.
Keywords: Antibiotic susceptabilty test; Bacterial fluorescence; Flow cytometry; Yersinia pestis.