Viable but non-culturable and persistence describe the same bacterial stress state

Bacteria are often thought of as having two dormant phenotypes: the viable but non-culturable (VBNC) state and the persister state. Here we investigate the relatedness of the two stress-induced phenotypes at the single-cell level and examine cell morphology and quantify cell resuscitation. Using the classic starvation conditions to create VBNC cells, we found that the majority of the remaining Escherichia coli population are spherical, have empty cytosol and fail to resuscitate; however, some of the spherical cells resuscitate immediately (most probably those with dense cytosol). Critically, all the culturable cells in this starved population became persister cells within 14 days of starvation. We found that the persister cells initially are rod-like, have clear but limited membrane damage, can resuscitate immediately and gradually become spherical by aging. After 24 h, only rod-shaped persister cells survive, and all the spherical cells lyse. Both cell populations formed under the VBNC-inducing conditions and the persister conditions are metabolically inactive. Therefore, the bacterial population consists of dead cells and persister cells in the VBNC-inducing conditions; that is, the non-lysed particles that do not resuscitate are dead, and the dormant cells that resuscitate are persister cells. Hence, ‘VBNC’ and ‘persister’ describe the same dormant phenotype.