Sociale interacties en antimicrobiële tolerantie in multispecies biofilmen uit de brouwerijsector

Micro-organisms predominantly live within dense, surface-associated communities, called biofilms. Moreover, most natural biofilms consist of multiple species that interact with each other and thereby influence one another. One hallmark of multispecies biofilms is their increased tolerance towards antimicrobial treatment. Consequently, multispecies biofilms still cause significant problems in industrial environments. A better understanding of the origin of the increased tolerance in these biofilms can contribute to the development of new anti-biofilm strategies. Therefore, the goal of this PhD research was to characterize the ecology and evolution behind this increased tolerance. To maximize the industrial application potential of our results, all research was conducted using multispecies biofilm models that were based on brewery biofilms. In a first step, microbial contamination in breweries was investigated by analyzing biofilm samples that were collected from three breweries in Belgium. Our results showed that the currently used disinfection methods are not sufficient to remove microbial contamination. Furthermore, we identified several species that could be found in multiple samples and were also isolated from biofilm samples in previous studies. Based on these samples, two types of biofilm models were constructed, that will be used for further research. Undefined models were grown directly from the complete biofilm samples and therefore, the exact species composition was not known. In contrast, defined models were set up by combining a fixed number of species that were isolated from the same sample. Next, a screening of known monospecies biofilm inhibitors was performed to identify compounds that are active against multispecies brewery biofilms. Hereto, undefined models were used and sulfathiazole was identified as the inhibitor with the most broad-spectrum activity. In a next step, the properties of the multispecies biofilms were further analyzed by investigating the inter-species interactions in 12 defined models. Based on the differences in growth of each species in mono- and multiculture, interactions were classified as competitive or cooperative. In agreement with previous studies, mainly competitive interactions were found. Subsequently, the antimicrobial tolerance of multispecies biofilms was investigated by adding sulfathiazole to three defined biofilm models. In all models, the tolerance was increased in the multispecies biofilm, compared to monospecies biofilms. In two of the models, the increased tolerance could be attributed to a reduction in competitive interactions. Strong competitors in these models were more sensitive to sulfathiazole than the suppressed species and a reduction in growth of the strong competitors consequently caused the suppressed species to bloom. The result was a lower percentage inhibition of the latter species and an overall increase in tolerance in the multispecies biofilm. In a final model, that consisted of a duo-species biofilm of Pseudomonas and Raoultella, the increased tolerance of Pseudomonas could not fully be explained by a reduction of competitive interactions. Here, the presence of a competing species directly enhanced the inherent tolerance of Pseudomonas to antimicrobial treatment. In a final part of this research, the increased tolerance of Pseudomonas in the duo-species biofilm model mentioned above was further characterized. This tolerance was not dependent on the physical presence of Raoultella and was therefore most likely caused by a secreted product. Additionally, resistance development within this duo-species biofilm was monitored in an evolution experiment of 80 days. The growth of Pseudomonas increased in the duo-species biofilm, while this species could not develop resistance to sulfathiazole in monoculture. Moreover, Pseudomonas species that were evolved for 36 days in the treated duo-species biofilm acquired a resistance, that was no longer dependent on the presence of Raoultella. In conclusion, our results provide a number of important new insights in the increased tolerance in multispecies biofilms. Firstly, we showed that the commonly observed competitive interactions in naturally occurring multispecies biofilms lie at the basis of the frequently reported increased antimicrobial tolerance of multispecies biofilms. Secondly, we found that the presence of competing species can also directly enhance the inherent tolerance of microbes to antimicrobial treatment. Finally, we observed that an initially small increase in tolerance of a species under the influence of the presence of other species, can lead to the evolution of a completely resistant species.status: publishe