Mechanisms of Resistance against Hop Beta Acids in Listeria monocytogenes

Promovendus/a: Maarten Goedseels

Promotor(en): Prof. dr. ir. Christiaan Michiels

Have you ever wondered why pregnant women are advised to avoid consuming certain cheeses, raw milk and some meat or fish products? This is because these products might be contaminated with the bacterium Listeria monocytogenes. While this name does not sound familiar to most people, the effects of the infection caused by these bacteria, listeriosis, are notorious. Fortunately, thanks to modern preservation techniques and strict hygienic measures in the food industry, infections with Listeria are infrequent. Nowadays, as people want healthier and more natural food options, preferably without chemical preservatives, new challenges in maintaining the microbiological safety of these products have risen. This has driven scientists to search nature for substances with antimicrobial properties to reduce the use of traditional food preservatives. Unfortunately, these natural alternatives often must be used in high amounts to be as effective as conventional preservatives. Therefore, it is crucial to understand how these natural compounds work, so they can be effectively implemented in novel food preservation strategies.

One promising source of natural antimicrobial compounds is the hop plant, known scientifically as Humulus lupulus. The flowers from the female plant have been used in beer brewing for several centuries already, not just for their flavour but also for their ability to prevent the beer from spoiling. Nowadays, we know that this latter effect can be attributed to certain molecules called hop bitter acids. Some of these bitter acids, including a group of molecules called the hop beta acids, have an exceptionally strong activity against certain bacteria, including Listeria monocytogenes. In this PhD project, we investigated why these hop beta acids have such a strong antimicrobial activity against Listeria. To answer this question, we chose a strategy in which we first tried to make the bacteria resistant to the hop beta acids to subsequently deduce the underlying resistance mechanisms and (elements of) the antimicrobial mode of action of the hop beta acids.

By repeatedly exposing Listeria to low amounts of hop beta acids, we were able to isolate three mutants that became resistant to the molecules. Two of these mutants had defects in a gene called mprF. Typically this gene helps protect bacteria against certain antimicrobials by adding positive charges to their cell surfaces. However, we found that for hop beta acids the opposite is true: fewer positive charges on the cell surface increases the resistance of the bacteria against the compounds, but simultaneously makes them more sensitive to other antimicrobials. The third mutant became resistant to the hop beta acids through another mechanism. It had a mutation in a gene called rex, which typically regulates other genes that are involved in the energy metabolism of the bacteria. The increased resistance of the third mutant could be attributed to cytochrome bd, which is one of the complexes regulated by Rex that helps the bacteria in energy production and dealing with some stressors.

By combining what we learned from these mutants with the research of other scientists, we proposed that hop beta acids have such a strong antimicrobial activity against Listeria because they target the cell in at least two ways. Firstly, they have a proton ionophoric effect, which means that they can carry protons into the bacterial cells and release them inside. This increases the acidity inside the cell and disrupts its energy production. Secondly, the hop beta acids can cause oxidative stress and deplete essential ions inside the cell by forming complexes with these ions and by facilitating damaging chemical reactions.

These findings highlight that the strong antimicrobial activity of hop beta acids is caused by more complex mechanisms than previously thought. This research provides new insights into how we could use hop beta acids and similar compounds as effective natural preservatives, potentially improving food safety and extending the shelf life of products without relying on synthetic chemicals.