Shining a light on antibiotic selection: optimised live/dead fluorescence spectrometry for rapid antimicrobial susceptibility testing

Antibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. We propose to use live/dead spectrometry as a rapid alternative to culture-based techniques through application of the LIVE/DEAD® BacLightTM Bacterial Viability Kit. We have developed a spectroscopic device (Optrode) to measure fluorescence from SYTO 9 and propidium iodide stained cells that can be used to enumerate the bacterial load. We propose a procedure using the Optrode that will take bacteria in a clinical sample, challenge with a panel of antibiotics, and measure live/dead ratios to determine the best bactericidal choice. Using calibration data we optimised the live/dead spectrometry protocol outlined in the kit instructions, improving upon media selection for growth and staining, and analytical parameters. We applied the optimised methodology to detect live and dead Escherichia coli in populations challenged with ampicillin. Killing was detected by the Optrode in near real-time when E. coli was treated with ampicillin and stained with SYTO 9 and/or PI. Following on from the promising results generated with ampicillin, live/dead spectrometry of ampicillin challenged cells was characterised in terms of antibiotic concentration, growth phase, and susceptibility to treatment for each treatment time. The generated data demonstrated that reliable detection of E. coli knockdown by ampicillin using live/dead spectrometry requires log phase cells challenged with a suitable concentration for a particular treatment time.