‘Viable but non‐culturable cells’ are dead

Abstract

Summary Most bacteria lead lives of quiet desperation, so they sleep. By sleeping, bacteria survive ubiquitous stress, such as antibiotics, and can resuscitate to reconstitute infections. As for other nearly universal and highly regulated processes such as biofilm formation, in persistence, a small population of cells have an elegantly‐regulated pathway to become dormant. By inactivating their ribosomes, persister cells sleep through stress and resuscitate once (i) the stress is removed, (ii) nutrients are presented and (iii) ribosome content reaches a threshold. During stress, cells often become spheroid and die, becoming hollow, membrane‐enclosed vessels. How cellular content is lost is unclear, but it is obvious that these ‘cell shells’ are dead; i.e., ‘There's no there there’. Critically, due to their intact membranes, the shells appear with membrane‐impenetrant stains as ‘viable’ particles. Unfortunately, the microbiology field of ‘viable but non‐culturable cells’ (VBNCs), though important for demonstrating the existence of dormant bacteria as a result of myriad stress states, has often mistaken these non‐viable shells as viable particles that mysteriously may be reborn, when an appropriate incantation is made. We argue here, based on experimental data, that if resuscitation occurs, it is the persister (always‐viable) cell population that revives, rather than the cell husks, which are dead.

What do we mean by viability in terms of ‘viable but non‐culturable’ cells?

“…Song and Wood assumed that if cells lost their ability to grow or form visible colonies on media, they were not viable, i.e. dead (Baquero and Levin, 2021;Song and Wood, 2021). This begs the question about cells that produce only limited growth, i.e.…”

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

“…We want to critically reply to the recent opinion article by Song and Wood (2021), claiming '"Viable but nonculturable cells" are dead'.…”

“…The paper of Song and Wood (2021) is helpful as it demonstrates how important definitions are: 'A bacterial cell in the VBNC state may be defined as one which fails to grow at the routine bacteriological cultivation conditions under which it would normally grow, but which in fact is alive and has still metabolic activity' (Oliver, 2010). Unfortunately, such a definition is missing in the opinion paper.…”

“…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. The same criticism can be applied for the publication Kim et al (2018) that represents the main source of data on which the opinion article by Song and Wood (2021) relies. In the paper by Kim et al (2018), the definition of VBNC used in table 1 is equally wrong.…”

“…If the title and conclusions of the article written by Song and Wood (2021) were true, all cells that are dormant and resuscitable would be cultivable and no non-culturable resuscitable cells would exist, i.e. there would be no state where cells survive by reducing their metabolism and completely losing their culturability.…”