Abstract
Rapid antimicrobial susceptibility testing is needed to reduce prescription of inappropriate antibiotics. A rapid alternative to standard culture-based testing is to determine reductions in cell viability using the LIVE/DEAD® BacLightTM Bacterial Viability Kit. We optimised the kit protocol for this application, focusing on simplifying the process by minimising the steps involved and on determining the optimal analytical parameters for fluorescence measurements from the dyes SYTO 9 and propidium iodide (PI). We demonstrate that for our experimental system, the intensity of emissions should be integrated from 505–515 nm for SYTO 9 and 600–610 nm for PI, and the proportion of live cells calculated from a new dye ratio formula, termed the adjusted dye ratio. We show that the pre-staining washing step is not necessary if a non-fluorescent growth media is used; however, staining must be done for each sampling as prolonged exposure to the dyes negatively impacts cell viability. The optimised methodology was able to reproducibly detect reductions in culture viability when the proportion of live cells in a sample of 1 × 108 cells/ml fell below ∼50% live in a media that supports the growth required for detecting antibiotic killing. Finally, we show that the interaction of fluorescence emission spectra from SYTO 9 and PI stained Escherichia coli cells is influenced by the proportion of dead cells in a sample. The excitation of PI by SYTO 9 was found to occur in populations containing sufficient numbers of dead cells (>25%), whereas in populations with low numbers of dead cells the dye interaction was additive in regard to red emissions, indicating that these dye interactions may offer another dimension to live/dead analysis. Fluorescence measurements from samples established according to the optimised protocol can be taken using a flow cytometer, spectrofluorometer, microplate reader, and the Optrode, a fibre-based spectroscopic system developed at the University of Auckland.
Highlights
Rates of antimicrobial resistance (AMR) are an increasingly significant health burden, in bacteria that cause common infections (WHO, 2014, 2017)
While the limit of detection (LoD) for live cells does not extend to a 99.9% reduction in viable cell plate counts, as required for determination of bactericidal activity, the results demonstrate the feasibility of quantifying differences in the viability of E. coli populations using the optimised methodology
The LIVE/DEAD R BacLightTM Bacterial Viability Kit can be applied to E. coli MG1655 in minimal A salts medium with 0.2% glucose to detect viability on samples that did not require washing before staining
Summary
Rates of antimicrobial resistance (AMR) are an increasingly significant health burden, in bacteria that cause common infections (WHO, 2014, 2017). A key strategy to address the problem of AMR is improved infection control, of which fast and reliable diagnostics is an important aspect. Antimicrobial susceptibility testing (AST) is used to determine the sensitivity of bacteria to potential antibiotic treatments (Jorgensen and Ferraro, 2009). Culture-based techniques are typically used, where live cells grow on bacteriological media while dead cells do not (Berney et al, 2007; Jorgensen and Ferraro, 2009). Culture-based methods are slow [typically obtained in two to five days (Lagier et al, 2015)], which can lead to prescription of an inappropriate antibiotic in a clinical setting due to urgency in treatment (WHO, 2014). Faster diagnostics that can inform antibiotic choice may circumvent this problem
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