How dead is dead? Viable but non‐culturable versus persister cells

[...]VBNC cells are determined using multiple viability criteria such as intracellular enzyme activity, cell membrane potential, ATP content, ribosomal activity as determined by FISH, protein synthesis, EMA- and PMA-PCR, CTC-FM or D2O-labelled Raman spectroscopy (e.g. Oliver 2010, Dwidjosiswojo et al., 2011, Alleron et al., 2013, Guo et al., 2019, Wideman et al., 2021), optimally determined simultaneously at the cellular level. Even regain of culturability was observed at some time points only in combination with specific human cell lines (Dietersdorfer et al., 2018) but never with the nutrient medium alone. [...]regain of culturability of VBNC Legionella cells in co-culture with amoebae was demonstrated several times by other authors (Epalle et al., 2015; Garcia et al., 2007). In order to determine the complete loss of culturability, copper-exposed cells of P. aeruginosa in the VBNC state were membrane filtered, washed, and the membranes were incubated on agar, but no growth was observed, indicating that all cells were non-culturable and that resuscitation was not the result of regrowth of an (even minute) subset of culturable cells (Wingender et al., unpublished information). Listeria monocytogenes remained cytotoxic even in the VBNC state (Wideman et al., 2021). [...]different species may behave differently and such a general conclusion as in this article is – according to Sir Karl Poppers falsification theory – not permissive.