Periodic Reporting for period 4 - COMBAT (Clearance Of Microbial Biofilms by Advancing diagnostics and Therapy) Reporting period: 2021-11-01 to 2022-04-30 Summary of the context and overall objectives of the project Biofilm-associated infections affect millions of people and are a leading cause of death and disability. With progress of medical sciences, more and more indwelling devices for the purpose of medical treatments and foreign body implants are applied. Infection continues to be a major complication of their use. Many efforts to decrease the burden of biofilm-associated infections have been made, however there is still no anti-biofilm compound in clinical use.Many of today´s antibiotics are very effective in the treatment of bacterial infections. However, their use in the successful eradication of biofilm-associated infection relies on our ability to overcome biofilm tolerance. In this project, we managed to obtain new knowledge on the mechanisms of biofilm tolerance of the gram-negative pathogen Pseudomonas aeruginosa. Our results on how bacterial metabolism and pH homeostasis impact the bacterial tolerance profiles can be exploited to improve current diagnostics and to develop more effective therapeutic intervention strategies for the control of biofilm-associated infections. In contrast to the application of therapeutics that kill biofilm-grown cells, those therapeutic interventions will include the sensitization of biofilm-grown pathogens to the effects of common antibiotics. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We have used extensive microbiological and bioinformatic expertise combined with access to genetic and phenotypic information of a high number (>400) of relevant biofilm-forming P. aeruginosa isolates to unlock the potential of microbial genomics. We found that biofilm tolerance is highly variable between individual strains and that the tolerance phenotype depends on the antibiotic; some antibiotics are more effective than others in killing biofilm-grown cells. Tolerance was also found to be independent on the resistance profile under planktonic growth conditions, and independent on the structural properties of the biofilms. However, bacterial tolerance was impacted by the metabolic status of the bacterial cells. By the use of extensive phenotype-genotype correlation studies and -omics based profiling of antibiotic tolerant versus non-tolerant bacterial isolates, we uncovered that changes in pH homeostasis determine tolerance phenotypes. We generated mutant strains and manipulated the bacterial pH homeostasis by interfering with the membrane potential to demonstrate the link between bacterial metabolism and the tolerance profile. Our results indicate that a therapeutic interference with bacterial metabolism can lead to a reversion of the biofilm tolerance phenotype, so that biofilm-grown bacteria become accessible towards conventional antimicrobial therapy. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Our results imply that biofilm-grown bacteria avoid antibiotic killing and become tolerant by counteracting intracellular alkalization through the adaptation of metabolic and transport functions. The link of biofilm tolerance with bacterial metabolism is interesting and opens up unique and novel possibilities to fight biofilm tolerance. Abrogation of antibiotic tolerance by interfering with the cell’s bioenergetics promises to pave the way for successful eradication of biofilm-associated infections. In this context, we are currently working on repurposing of already approved drugs as biofilm-sensitizing agents. This has the potential to accelerate the introduction of new treatments for recalcitrant biofilm-associated infections into the clinic.