The VBNC state and resistance to environemental stress factors.

As a common method to survive harsh conditions bacteria enter a viable but non culturable state (VBNC). In this state metabolic activity is at a minimal level, but as soon at favourable conditions return they revert to a normal growth. Vibrio vulnificus is recognised as one of the major sea-food associated pathogens, and it is well known that when water temperatures are below 15 ^oC they enter a VBNC state. While it is generally accepted that bacteria enter this VBNC state, the effects this has on resistance to environmental stress factors is less well known. Nowakowska and Oliver (2013) set out to document the effect of several environmental factors on the normal culturable and VBNC state of V. vulnificus. 

For this study they used two different V. vulnificus strains: C7184 as a clinical strain and JY170 as an environmental strain. Cultures were either used for direct exposure to environmental stresses or stored at 5 ^oC to induce the VBNC state, after which they were exposed to the stress factors. Immediately before and after exposure to the challenges a viability assay was conducted , by determining cell membrane integrity, to determine the effects of the stresses on the culture. For cells from the VBNC state that were exposed to the stress factors, they resuscitated by culturing overnight at 22 ^oC and then plated onto agar to determine viability. Stress factors applied were: Temperature, Oxidative stress, Osmotic stress, pH, ethanol, antibiotic and heavy metals. 

Overall results indicate that cells in the logarithmic growth phase appear to be highly susceptible to the tested environmental stresses, but that cells from the VBNC state appear to be mostly resistant. Both the clinical and environmental strains lost culturabillity when exposed to temperatures over 42 ^oC, but VBNC cells were able to recover after overnight resuscitation. The same was found to be the case for cells exposed to ethanol. When exposed to hypersaline environments log phase cells for both the clinical and environmental strain lost culturability, but interestingly the VBNC cells for the clinical were found to be highly sensitive, where as the environmental strain was found to be significant resistance. A similar result to the salinity stress was found in response to oxidative stress and both high and low pH, with the environmental VBNC cells exhibiting a higher tolerance compared to the other cultures. When cultures were exposed to antibiotics and heavy metals it was found that yet again the VBNC cells exhibited a higher level of resistance. Though it appears that there is a difference depending on the heavy metal compound, with several copper containing compounds being resisted but others causing cell death.

This study is of interest as many of the cleaning methods used in the food industry rely on cleaning with highly acidic or alkaline substances, pasteurisation, or similar methods. This would suggest that these cleaning methods are ineffective. While the growing cells may be removed, the VBNC cells may be able to return to a normal logarithmic growth and cause infection even after supposed cleaning and sterilisation. Overall this means that while we think we may be safe and the food industry is clean, we may still be subject to many dangerous pathogens. 

Nowakowska, J., Oliver, J., D.. (2013). Resistance to environmental stresses by Vibrio vulnificus in the viable but nonculturable state. FEMS Microbial Ecology. 84, 213-222.