Uncovering the Molecular Basis of Formation of Viable But Non-Culturable Cells

Vibrio parahaemolyticus is a bacterium that is present in the marine environment and can be found in seawater, shellfish (such as oysters and mussels) and in crustacea (such as crab). This bacterium is the leading cause of seafood associated gastroenteritis worldwide. The bacterium can be destroyed during the cooking process, thus infection is generally associated with eating raw shellfish or cooked seafood products that have been cross-contaminated by raw shellfish or contaminated water. V. parahaemolyticus infections peak in the summer seasons, when sea temperatures are optimum for its growth. In the last 10 years the number of V. parahaemolyticus outbreaks has increased worldwide and has been as a result of rising sea temperatures. Climate change, globalisation and other drivers have also made Europe a hot spot for emerging infectious diseases including infections by V. parahaemolyticus. At present, detection of V. parahaemolyticus is not required under EU Food Hygiene legislation for testing of shellfish harvesting areas and ready to eat seafood products. Furthermore, disease associated with V. parahaemolyticus is not notifiable in the EU but in recent years there have been a number of outbreaks associated with contaminated seafood in Europe including Spain, Italy and Norway that have begun to change the significance of this pathogen in Europe.

In the past it has been shown that when bacteria were incubated in sea water, the cells remained 'alive' but could not grow on culture media. However, they sometimes retain the ability to cause disease. These bacteria have been called 'viable but non-culturable' (VBNC) cells. The VBNC cells allow the bacteria to survive, until more favourable conditions arise. Cells in the VBNC state have low metabolic activity but, following environmental stimuli such as temperature upshift or nutrient supplementation, they can 'resuscitate' and restore their ability to grow on media. VBNC cells are not just important in a medical context because of their capacity to remain virulent and resuscitate in favourable conditions but they are also important in food safety. If VBNC cells are present in food samples and cannot be detected by conventional techniques, then the number of bacteria in food samples could be underestimated.

In this project we will investigate the potential risk of V. parahaemolyticus VBNC present in seafoods and the risk they may pose to the seafood industry. We will also try to understand what genes present in V. parahaemolyticus cause this bacterium to enter the VBNC state and help in its resuscitation. Our aim is to help the seafood industry come up with ways to help manage the risk VBNC cells pose to the industry.

Vibrio parahaemolyticus is a human pathogen and globally the leading cause of seafood-associated gastroenteritis. A recent report indicates that pathogenic Vibrio species pose one of the greatest risks to public health in the European Union in the coming years because of the likelihood that climate change will result in an increase in the incidence of disease. This research project looks at the ability of V. parahaemolyticus to form viable but non-culturable (VBNC) cells when exposed to stressful conditions. VBNC cells are viable or metabolically active, but are unable to form colonies on standard culture media. Following environmental stimuli, such as temperature upshift, some VBNC cells can 'resuscitate' restoring their ability to grow on media. The ability for VBNC cells to go undetected by conventional microbiological practices could lead to an underestimation of total viable cells in environmental and clinical samples. Furthermore, their capacity to retain virulence potential and their ability for renewed metabolic activity means the VBNC state in V. parahaemolyticus may pose a risk to human health. We have developed robust models to generate V. parahaemolyticus VBNC cells in the laboratory and shown that on the basis of the ability of the VBNC cells to grow on laboratory media, metabolic activity, cell shape and the ability to grow and cause disease in Galleria mellonella, we can identify distinct types of VBNC cells. In this project we will identify genes which play roles in VBNC formation and characterise the proteomic makeup of different types of VBNC cells. This will allow us to understand the molecular processes that lead to VBNC cell formation and identify markers for VBNC cells. In parallel we will investigate the formation of VBNC cells in artificially infected oysters. Our overall aim is to enable the seafood industry to develop intervention strategies that will allow the elimination or control of V. parahaemolyticus in the food chain.

Our science will lead to tangible and relevant advice to help develop intervention strategies that will in turn lead to the prevention and/or control of V. parahaemolyticus in the food chain. The main aim of our impact activities will be to engage and collaborate with our industrial partners and government bodies such as Defra to develop and help implement these intervention and control strategies into policy. The research from this project will directly impact seafood industry in the UK. We will be working closely with members of the seafood industry to ensure that we translate our knowledge in order to help develop strategies to reduce and control bacterial contamination of seafood.

The research from this project will be of value to policy makers such as Defra, and its agency called Cefas (Centre for Environment, Fisheries and Aquaculture Science). Cefas are the designated European Reference Laboratory for shellfish monitoring and play a large role in policy advice on seafood safety to other EU member states and UK based shellfish farmers. The information from this project may lead to changes in shellfish farming or processing practice which could have a significant impact on infection control.
The information from this project will be helpful to those working on V. parahaemolyticus in seafood in South East Asia and USA where this pathogen is the leading cause of seafood gastroenteritis. This will include agencies such as the US Food and Drug Administration who are responsible for seafood safety in the USA.