Enhancing the anti-biofilm activity of 5-aryl-2-aminoimidazoles through nature inspired dimerisation.

The increased tolerance of biofilms against disinfectants and antibiotics has stimulated research into new methods of biofilm prevention and eradication. In our previous work, we have identified the 5-aryl-2-aminoimidazole core as a scaffold that demonstrates preventive activity against biofilm formation of a broad range of bacterial and fungal species. Inspired by the dimeric nature of natural 2-aminoimidazoles of the oroidin family, we investigated the potential of dimers of our decorated 5-aryl-2-aminoimidazoles as biofilm inhibitors. A synthetic approach towards 2-aminoimidazole dimers linked by an alkyl chain was developed and a total of 48 dimers were synthesized. The linkers were introduced at two different positions, the N1-position or the N2-position, and the linker length and the substitution of the 5-phenyl ring (H, F, Cl, Br) were varied. Although, no clear correlation between linker length and biofilm inhibition was observed, a strong increase in anti-biofilm activity for almost all N1,N1'-linked dimers was obtained, compared to the respective monomers against Salmonella Typhimurium, Escherichia coli and Staphylococcus aureus. The N2,N2'-linked dimers, having a H- or F-substitution, were also found to show a strong increase in anti-biofilm activity compared to the respective monomers against these three bacterial species and against Pseudomonas aeruginosa. In addition, the obtained growth measurements suggest a broad concentration range with specific biofilm inhibition and no effect on the planktonic growth against Salmonella Typhimurium and Pseudomonas aeruginosa.

Inhibiting bacterial cooperation is an evolutionarily robust anti-biofilm strategy.

Bacteria commonly form dense biofilms encased in extracellular polymeric substances (EPS). Biofilms are often extremely tolerant to antimicrobials but their reliance on shared EPS may also be a weakness as social evolution theory predicts that inhibiting shared traits can select against resistance. Here we show that EPS of Salmonella biofilms is a cooperative trait whose benefit is shared among cells, and that EPS inhibition reduces both cell attachment and antimicrobial tolerance. We then compare an EPS inhibitor to conventional antimicrobials in an evolutionary experiment. While resistance against conventional antimicrobials rapidly evolves, we see no evolution of resistance to EPS inhibition. We further show that a resistant strain is outcompeted by a susceptible strain under EPS inhibitor treatment, explaining why resistance does not evolve. Our work suggests that targeting cooperative traits is a viable solution to the problem of antimicrobial resistance.

Biofilm Bacteria Use Stress Responses to Detect and Respond to Competitors.

Bacteria use complex regulatory networks to cope with stress, but the function of these networks in natural habitats is poorly understood. The competition sensing hypothesis states that bacterial stress response systems can serve to detect ecological competition, but studying regulatory responses in diverse communities is challenging. Here, we solve this problem by using differential fluorescence induction to screen the Salmonella Typhimurium genome for loci that respond, at the single-cell level, to life in biofilms with competing strains of S. Typhimurium and Escherichia coli. This screening reveals the presence of competing strains drives up the expression of genes associated with biofilm matrix production (CsgD pathway), epithelial invasion (SPI1 invasion system), and, finally, chemical efflux and antibiotic tolerance (TolC efflux pump and AadA aminoglycoside 3-adenyltransferase). We validate that these regulatory changes result in the predicted phenotypic changes in biofilm, mammalian cell invasion, and antibiotic tolerance. We further show that these responses arise via activation of major stress responses, providing direct support for the competition sensing hypothesis. Moreover, inactivation of the type VI secretion system (T6SS) of a competitor annuls the responses to competition, indicating that T6SS-derived cell damage activates these stress response systems. Our work shows that bacteria use stress responses to detect and respond to competition in a manner important for major phenotypes, including biofilm formation, virulence, and antibiotic tolerance.