Bioelectrochemical detection of viable but non-culturable bacterial cells on screen-printed carbon electrodes

Bacterial cells in a viable but non-culturable (VBNC) state are metabolically active, but cannot be cultivated using a routine growth medium, which poses a challenge to identifying VBNC contamination in food and the health sector. Non-growth assays for VBNC identification based on membrane integrity and metabolic monitoring are either costly or lead to ambiguous results. Nucleic acid (DNA and RNA) amplification techniques are effective, but their higher cost and complexity prevent routine applications. Bioelectrochemical assays might be a viable alternative for VBNC detection, due to their low cost and rapid analysis time. However, conventional bioelectrochemical methods, in which electrodes are polarized at constant electrochemical potential, are not suitable for monitoring planktonic non-growing cells like VBNC. To circumvent this issue, culturable and VBNC Pseudomonas aeruginosa cells were embedded in hydroxyethyl cellulose (HEC) coating and exposed to alternated cathodic and anodic potentials for a short-time. The resulting current output was interpreted in terms of charge/discharge of the bacterial membrane at the polarized electrode, which is a proxy for bacterial viability. P. aeruginosa cells were induced into the VBNC state by either UV-C or NaOCl. In the presence of 5 mM K₃[Fe(CN)₆] and 20 mM glucose, the current output correlates inversely with the VBNC cells concentration. For cells inactivated by heat or 4% paraformaldehyde, the current output was not significantly different from the blank electrode, indicating the ability of the proposed bioelectrochemical method to detect changes in cellular viability before the loss of culturability.