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Abstract:

Bacteria do not live in isolation but in communities where several kinds of interactions occur, from mutualism to competition. These communities can be composed of bacteria from a single species or from multiple species, sometimes coexisting with non-bacterial organisms such as yeasts. These kinds of interactions define several behaviors of the bacteria in their communities and are key to survival in harsh environments. A specific form of these communities are bacterial biofilms. Biofilms are bacterial conglomerates in an interphase that are protected by a layer of Extracellular Polymeric Substances (EPS). This makes biofilms especially resistant to antimicrobials and desiccation, thus making them a problem in clinical settings, where classical antibiotics and disinfectants are unable to eliminate biofilms. Thus, alternatives to classical antimicrobials are needed. A first option is looking into known natural products endowed with antibiofilm activity and study how their chemistry can be modified to enhance this antibiofilm activity. Ideally, these compounds should be able to disrupt or prevent biofilm formation without killing the bacterial cells, since it has been hypothesized that non-lethal inhibition of virulence factors generates less selective pressure towards antimicrobial resistance. In this regard, tannins emerge as an alternative for their proven record as bioactive molecules. In the first chapter of this thesis, we show that tannins can be fine-tuned to modify their potency, antimicrobial spectrum, as well as biofilm specificity. A second approach is looking into specific mechanisms to combat biofilm formation in a way that reduces the probability of antimicrobial resistance. This can be done by taking advantage of cooperation in monospecies biofilms via public goods. Public goods are bacterial-produced substances that: 1) are beneficial to the biofilm community, 2) are costly to produce, and 3) are exploitable. Public goods are particularly interesting targets for antibiofilm drugs, because resistance against these drugs is hypothesized to be counter-selected. The reason is that a resistant bacterium that has regained public good production will share the public good with the sensitive bacteria around, but it will not share the energy cost to produce the public good. The sensitive bacteria therefore have a net advantage and outcompete the resistant bacteria. This hypothesis was proven in Salmonella Typhimurium, where a 2-aminoimidazole derivative was able to keep its activity against the production of public exopolymeric substances in Salmonella biofilms after 40 days of treatment, even inducing a phenotype that produces less biofilm than the parental strain. In this thesis we show that this reduction in biofilm phenotype could be explained by mutations in the biofilm regulator RpoS and the β-subunit of RNA polymerase that are associated with a reduced biofilm biomass formation, but an increased bacterial biofilm population, [HS1] [XV2] which can be due to an inhibition of the transition from exponential phase metabolism to stationary phase metabolism. A third approach is to interfere with the competition tools of multispecies bacterial biofilms. In a mixed species biofilm, Pseudomonas aeruginosa usually outcompetes Staphylococcus aureus because of its production of soluble toxins. However, S. aureus is able to defend via a stress response system called SigB that induces biofilm formation and therefore potentially also enhances antimicrobial tolerance. We tested if inhibition of the SigB stress response system could reduce biofilm tolerance of a mixed species biofilm of S. aureus and P. aeruginosa; however, it was not possible to demonstrate so. It could only be determined that SigB increased biofilm tolerance and bacterial attachment in monospecies biofilms but not in mixed species biofilms. This was due to the weak level of protection that SigB offered against P. aeruginosa.