Bacterial dormancy: a subpopulation of viable but non-culturable cells demonstrates better fitness for revival

Abstract

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two key subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. The two subpopulations displayed different abilities for revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) has also revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. The proteins that were significantly up and down regulated between the different VBNC populations were determined and significantly regulated proteins were assigned into 23 functional groups, the majority being included in metabolism functional categories. A lactate dehydrogenase (lldD) protein was significantly upregulated in all subpopulations of VBNC cells and is responsible for converting lactate to pyruvate. Deletion of the lactate dehydrogenase (RIMD2210633:{Delta}lldD) gene causes the cells to enter the VBNC state significantly faster than the wild-type, and exogenously adding lactate to VBNC cells aided resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:{Delta}lldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase plays a putative key role in regulating the VBNC state.