Implementatie van Nieuwe Anti-Biofilm Technologieën

The overuse and incorrect use of antibiotics has resulted in the spread of multidrug-resistant bacteria. Infections caused by these bacteria are difficult and sometimes impossible to treat. By 2050, roughly 10 million deaths globally are expected to result directly or indirectly from antimicrobial resistance. To put it into perspective, this is 8 times higher compared to deaths caused by diabetes or traffic incidents and equivalent to deaths caused by cancer1. Various alternative treatment and prevention strategies have been proposed. One possibility is targeting the virulence factors of bacteria with anti-virulence strategies. Virulence factors are not essential for bacterial growth but enable the pathogen to cause an infection. By targeting virulence factors, the pathogen becomes less virulent, and it possibly allows easier clearance of the infection. A sub-strategy is public good inhibition. Public goods are (i) costly, (ii) shared, and (iii) offer a benefit. Since they are shared, non-producers, we call cheaters, can thus exploit them. The most interesting aspect of public good inhibition is the theoretical selection against resistance. Indeed, if a bacterium becomes resistant against the public good inhibitor, it will resume production of the public good and pay an energy cost to do so. It will however share the public good with the sensitive cells surrounding it. In turn, they will also profit from the benefit of the public good without having a cost. As a result, they will have a net fitness benefit and outcompete the resistant cells. An example of a public good is the exopolymeric substances found in biofilms. Biofilms are complex, heterogenous, multicellular complexes encircled by a self-produced matrix. These biofilms can render bacteria 100 to 1000 times more tolerant to external stressors, including antimicrobial treatment. Moreover, due to the close proximity of cells, genes that result in antimicrobial resistance can more easily spread through the population. In this PhD, we used the 2-amino-imidazoles (2-AI) as inhibitors for biofilm formation in Salmonella Typhimurium and Staphylococcus aureus. In S. Typhimurium, this inhibitor was previously shown to decrease expression of the biofilm master-regulator, csgD2. Moreover, resistance against 2-AI treatment did not evolve after 40 days3. While we did not find resistance against the biofilm inhibition, we did observe mutations resulting in increased efflux activity. Therefore, we aimed to elucidate the reason for the selection of these mutations and their resulting phenotypes. The selection for these mutations was found to be due to a side-effect of the inhibitor that influenced bacterial growth. Increased efflux activity was then found to alleviate this growth-delaying side-effect. The next step was to create a 2-AI inhibitor that was more evolutionarily robust. This new inhibitor retained its anti-biofilm activity, while no longer affecting bacterial growth. Next, we wanted to study the mode-of-action of the inhibitor, as the activity of the inhibitor was shown to be dependent on the concentration and whether its coated or in solution. We hypothesized that the mode-of-action was twofold. The anti-biofilm activity, at low concentrations of the inhibitor, was due to some interactions of the inhibitor with the bacterial membrane, while the anti-planktonic activity was due to excess inhibitor penetrating the cell. While we did not confirm the anti-biofilm mode-of-action, we did find membrane permeabilizing activity at concentrations that coincide with anti-planktonic activity. Finally, we applied this inhibitor in two ways. First, we used the inhibitor as a coating and tested it in a rabbit model for fracture related infections (FRI) to evaluate whether biofilm inhibition could be a viable strategy to prevent and/or treat FRI's. No significant clearance of the infection was found in vivo. However, a trend was observed that showed better infection clearance when a prophylactic shot was used. In the second application, a glycyrrhizinic acid (GA) hydrogel was used as a carrier. First, we characterized the activity of this hydrogel in vitro and found that the antimicrobial activity of GA was pH-dependent. Afterwards, we created a GA-2‑AI hydrogel and characterized it in vitro and in vivo. While we did not find better clearance of the infection when both GA and GA-2-AI were present, surprisingly better bone healing was found in the presence of the GA hydrogel.