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 subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.
Highlights
Members of the Proteobacteria are reported to have the ability to form viable but non-culturable (VBNC) cells, a fundamental survival mechanism that allows bacteria to ‘hibernate’ or lay dormant until conditions become more favourable to support their growth [1,2]
Our results showed that two subpopulations (P1 and Population 2 (P2)) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods
The VBNC state can be induced in V. parahaemolyticus by low temperatures and salinity, secondly V. parahaemolyticus VBNC cells can be resuscitated by increasing the temperature in the medium and thirdly, the period of resuscitation of VBNC cells is well documented to be approximately 2 weeks after cells have become unculturable [8,11,15,16,17,18]
Summary
Members of the Proteobacteria are reported to have the ability to form viable but non-culturable (VBNC) cells, a fundamental survival mechanism that allows bacteria to ‘hibernate’ or lay dormant until conditions become more favourable to support their growth [1,2]. VBNC bacteria remain metabolically active albeit at a reduced capacity, but no longer form colonies on standard culture media [11,12,13]. Following environmental stimuli or permissible growth conditions, some VBNC cells can ‘resuscitate’, restoring their ability to grow on media [1,13]. The VBNC state has been well documented in Vibrio species and V. parahaemolyticus is a good model organism for studying VBNC cells for a number of reasons [14,15,16]. The VBNC state can be induced in V. parahaemolyticus by low temperatures and salinity, secondly V. parahaemolyticus VBNC cells can be resuscitated by increasing the temperature in the medium and thirdly, the period of resuscitation of VBNC cells is well documented to be approximately 2 weeks after cells have become unculturable [8,11,15,16,17,18]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
