One step ahead of microbial infections by disrupting cell communication of biofilm-forming bacteria

Author: Richard Bezemer, LabVision

AHV International develops, produces and markets solutions, which support and optimise the health of farm animals through their proactive action against bacterial invasions. The compounds extracted from plant extracts disrupt or break down the formation of biofilms. A microbial, cell culture and chemical-analytical lab have been set up to develop new products and research the precise action of these quorum sensing antagonists. Head of R&D Luc Grisez discusses the potential of these still relatively unknown fighters of bacterial invasions.

In bacteria, cooperation is in the genes. ‘As a bacterium alone, you are vulnerable to an army of host defence mechanisms. Moreover, it is not so easy to attach to the wall of, say, a gut and before you know it, you are carried away by a stream of body fluids. Under the evolutionary guise of standing strong together, bacteria in their millions are able to form a biofilm, which almost literally acts as a protective wall against external factors such as the host’s immune system, but also antibiotics. Moreover, such a biofilm can adhere extremely well and stays nicely where it is,’ Luc Grisez explains. He is head of R&D at AHV (Animal Health Vision) in Zwolle, which focuses on the development, production and sale of agents, which counteract or break down biofilms. Its focus is on animals, especially cows, pigs, goats, poultry and farmed fish for which there is a range of products on the market.

Brush properly

The majority of bacteria form biofilms. This can be in humans or animals, but also in environments and facilities where there are favourable growing conditions, such as on the inside of boilers and cooling towers, or on fishing nets. In humans, plaque is the most tangible biofilm (so brush well to remove it mechanically), but biofilm formation also occurs in intestinal, urinary tract and lung infections. Farm animals face similar infections, but there are also specific conditions such as mastitis (inflammation of the udder), which has a direct impact on milk production.

‘Under the evolutionary guise of together you are strong, bacteria are able to form a biofilm in their millions,’ he says.

Quorum sensing

To form biofilms, bacteria use an ingenious mechanism of cell-to-cell communication known as quorum sensing. The phenomenon, discovered in the 1970s, has not yet revealed all its secrets in detail. What is clear, however, is that a good number of bacteria must be present to trigger the formation of a biofilm. ‘If you have one bacterium, and it says of ‘I’m fine here, but I lack nutrients’, it has to start producing certain enzymes to get those nutrients. But chances are that this enzyme will drift away. So it is not energetically responsible to do that. However, if millions of bacteria are in close proximity to each other, then so much of that enzyme is produced that the nutrients can flow back and it does make sense to follow certain energetic pathways and recover the profit from that. A bacterium also produces intra- and intercellular signalling molecules that diffuse outwards. From a certain concentration, a quorum (a minimum necessary amount) is reached, so they are reabsorbed and induce expression of genes, which ensure formation of the biofilm and production of certain enzymes and toxins.’

Biofilm formation has about five stages. It starts with a rather loose attachment to host cells (absorption). After this, they adhere more strongly by forming a monolayer, which is effectively the beginning of colonisation. At this stage, a polymeric matrix layer is also formed, which protects the bacteria. After full growth of the biofilm (through cell division), part of the biofilm can detach to start a new cycle of biofilm formation.

Tackling biofilm

The biofilm not only provides an excellent barrier against the host immune system, but also gives antibiotics little chance of success. Bacteria in the biofilm can go into a kind of dormant state, in which they are metabolically barely active. In that state, where no cell division also occurs, they are up to 40x less sensitive to antibiotics. ‘Antibiotics are still widely used in farm animals. They are a good temporary solution for acute cases because they target the planktonic bacteria that are floating freely. But this does not eliminate the infection in the biofilm. Moreover, by directly interfering with bacteria in a biofilm, you create a greater chance of developing antimicrobial resistance,’ Luc says.

‘We don’t kill the bacteria, but we prevent them from establishing themselves or break down their establishment.’

Not so much an alternative to antibiotics, but rather a complement that makes antibiotics much less necessary, are the quorum sensing antagonists developed by AHV. ‘The action of these compounds is aimed at ‘tuning’ the metabolism of the bacteria. We do not kill the bacterium, but we prevent it from establishing itself or break down its establishment, the biofilm, so that it cannot express its virulence properties. This leaves it susceptible, not only to antibiotics, but also to the host’s immune system. That can then clean up the bacteria itself. Because we target the biofilm, which is the cause of the problem, we can ensure that the problem stays away for longer. Biofilms actually get in the way of effective antibiotic treatment, allowing infections to return. By removing the biofilm, that chance is much lower. This benefits the health of the animal, which thus has more energy to deal with stressors. In practice, that leads to a longer life of the animal and higher productivity.’

Preventive

Luc emphasises that antagonists are not drugs, as antibiotics are. ‘Our vision is that our solutions are used proactively. For this, we have developed animal-specific protocols, where the products are administered often orally as an additive to the feed or as a tablet. For example, a cow has fixed stress moments during lactation, such as at calving. This is when the risk of infection is higher. We can anticipate these moments with our products in the form of a proactive protocol that ‘cleans up’ the animal beforehand, removes the biofilm so the animal does not have to expend extra energy fighting an acute infection. Its own immune system can then fight it off just fine.’

Effective compounds

For the search for effective compounds, the plant world offers a seemingly inexhaustible source. ‘Plants are a logical source. They have no immune system and can only protect themselves from harmful actors by other chemical means. In the literature, we have found many suitable components or classes of compounds. But we also test in the lab the pathogens that interest us, such as Streptococcus, Campylobacter, E. coli and Salmonella, against plant extracts. A selection then comes out of that, based on which we put together a product. Bear in mind that it is often a combination of components. The mechanism of biofilm formation is bacteria-dependent, but the combinations of substances we use are widely applicable,’ says Luc.

Cold war

Quorum sensing has become a catch-all term for multiple mechanisms as a result of a lot of research in recent years. For example, there is now also evidence of an interaction between what a bacterium produces in terms of signalling molecules and its effect on the immune system. So there is also an effect between bacteria and host. That sequence of interacting systems can be thought of as a cold war at the bacterial level.

To also take those sequential effects into account, AHV has broadened its development focus, which was initially purely on biofilm. ‘We are also looking at the effects on immune modulation and stimulation, also in the context of reducing the inflammatory response. Furthermore, we are exploring the extent to which we can spare the non-harmful bacteria that, for example, help digest food in the gut. We are also looking at the integrity of the intestinal wall, which is linked to the condition of literally one layer of cells, which must remain intact. You can strengthen the bond between those cells, which benefits intestinal integrity and optimal function,’ Luc explains.

Besides searching for new substances, much work is also done in the labs to develop formulations. Initially, the products were mainly in the form of tablets, which is ideal for administration to a cow, but less so for chickens or fish. This should preferably be done through drinking water or feed. So how do you get the product in the right dosage; does it remain stable over a period of time?

Three labs

AHV has a BSL-2 microbial lab, a cell culture lab and a chemical-analytical lab, where five researchers are currently working. The microbial lab looks at the influences on specific bacteria. Do the products work the same way on different bacterial species, and how exactly does that work? This involves looking not only at the biofilm, but also at bacterial mortality, enzyme production and toxins.

Several cow and pig cell lines are grown in the cell culture lab for the study. The researchers look at the cytotoxicity of the products: what are safe doses at cellular level; at immuno-modulation: how do we stimulate the host’s immune response and develop methods for investigating the integrity of the intestinal wall, for example.

In the chemical-analytical lab, quality control of raw materials and AHV end products takes place first and foremost. But more and more work also goes into investigating what happens to components during production and administration, using LC-MS, among other methods. ‘For a formulation for salmon, we mix our product with fish feed. That is pelletised. That involves steam and high temperatures. The question is then whether the components remain active and available after that treatment. What is the degradation as a function of time? Can we find something back in the gut, in the blood?’, Luc gives as an example.

More water needed

As the product range expands and research intensifies, more and more water is also needed, both demineralised water and ultrapure water. Last year, AHV has already invested in a Purelab Flex 3 supplied by Veolia Water Technologies, which turns tap water into ultrapure water, used for the LC-MS, among other things. The desktop system provides a maximum flow of two litres of ultrapure water per minute.

For the demineralised water, which is sold in all three labs but has the autoclave as a major user, AHV is orienting itself towards a system that is easier to use than the existing one. That, in fact, works with filter cartridges, which have to be regenerated every so often, which, especially with the growing demand from cell culture and chemical analysis, is not convenient to work with. Veolia is in the race for that too, with a Chorus 3 RO system. However, the decision on this has not yet been made.