Stressed? Just enter a VBNC state!

Many different bacteria from several environments have the ability to enter a VBNC (viable but non-culturable) stage when under conditions which prove to be stressful to survive and function in properly. Included in the bacteria which are capable of this are a variety of human pathogens, as well as those which infect fish, sea urchins, and corals etc. The VBNC stage can be induced by temperature changes, changes in osmotic concentrations, oxygen levels, heavy metals and white light exposure.

When exposed to such conditions, cells show a significant amount of size reduction, major decreases in respiration rates and macromolecular synthesis, but ATP levels and membrane potential remain high, as well as remaining metabolically active. Seeing as VBNC cells cannot be cultured, alternative methods were required to prove that they were alive in this state. Reagents were commonly used which were designed to show the presence of an intact cytoplasmic membrane. A more recent approach to demonstrate the viability of these cells is to use a reverse transcriptase PCR, which detects gene expression. This is used because the half life of bacterial mRNA is ca. 3-5 minutes, so the continued gene expression by these non culturable cells shows their viability. It is the later method, amongst other molecular techniques that have put a stop to the disagreements surrounding the term ‘viable but non-culturable’.

When in the VBNC state, many pathogens have been observed to be capable of initiating infection, although not all investigations have reported this. In fact, some reports state that pathogens are avirulent in this state. However, the most likely case is that pathogens generally do not have the ability to initiate disease when in the VBNC state, but the virulence is retained and then initiated upon resuscitation from the VBNC state (This is often the case in biofilms).

The VBNC state must only beneficial in terms of survival if they are able to restore their metabolic activity and become culturable once more. It is difficult to prove this, as opposed to being able to regrow undetected, culturable cells present in the VBNC population. Induced VBNC and resuscitation in bacteria is most extensively studied in Vibrio vulnificus using temperature in vivo in calms, in situ in estuarine waters, and in vitro.

It has been shown that several higher organisms may be biological mediators of resuscitation from the VBNC state. For example, Legionella pneumophila entered the VBNC state following starvation and hypochlorite treatment, but was resuscitated once in the protozoans Acanthamoeba polyphaga and A. castellanii. Another development in the reactivation of dormant cells is that of the role of Rpfs (Resuscitation-promoting factors). Some Rpfs are peptidoglycan hydrolases which are involved in cell wall digestion, and therefore cell division. So peptidoglycan rearrangement appears to be prominent in VBNC states.

The exact role of VBNC states in bacteria is yet to be elucidated, but it is likely that the role varies amongst bacteria. The general understanding is that the VBNC state is activated to protect the cell from environmental stressors to allow their survival if and once the stress subsides. What is known is that the VBNC state is critical to the survival of cells, in particular, human pathogens, and possibly in their ability to produce disease. This is an important part of VBNC states as it creates a gateway of research into the reoccurrence of infections in humans, and in 2009, Epstein proposed that emerging from the VBNC state is a way of ‘sending out scouts to test the environment’ to ensure it is suitable to survive in, and in the case of a suitable environment, a signal would be sent out to resuscitate the remaining cells.

Oliver, J. D. (2009) Recent findings on the viable but non-culturable state in pathogenic bacteria. FEMS. 34: 415-425.