Tn-Seq-based identification of genes that play a role in antimicrobial tolerance of Pseudomonas aeruginosa aggregates

Microbial biofilms, whether surface-associated or in the form of aggregated microbes, cause a variety of persistent infections due to their inherent tolerance to antibiotic treatment. Current knowledge of the mechanistic basis of biofilm-associated antibiotic tolerance is fragmented, and the relative importance of previously identified factors remains unknown. We developed a Tn-Seq-based workflow that enables simultaneous assessment of the relative antibiotic tolerance of thousands of individual clonal bacterial aggregates, each established by a single transposon mutant. Using this genome-wide Tn-Seq analysis approach, we identified several factors and pathways that play a role in antimicrobial tolerance of Pseudomonas aeruginosa aggregates. The majority of these factors are important for aggregate-associated tolerance to specific antibiotics, whereas some factors play a role in aggregate-associated tolerance regardless of the class of antibiotic. This methodology provides a framework that can be adapted to investigate aggregate-associated tolerance for other bacterial species and in response to different antimicrobial agents and stressors.