Motility-independent formation of antibiotic-tolerant Pseudomonas aeruginosa aggregates

Pseudomonas aeruginosa is a bacterial pathogen that causes severe chronic infections in immunocompromised individuals. This bacterium is highly adaptable to its environments which frequently select for traits that promote bacterial persistence. A clinically significant temporal adaptation is the formation of surface- or cell- adhered bacterial biofilms which are associated with increased resistance to immune and antibiotic clearance. Extensive research supports that bacterial flagellar motility promotes formation of such biofilms whereupon the bacteria subsequently become non-motile. However, recent evidence supports that antibiotic-tolerant non-attached bacterial aggregates, distinct from surface-adhered biofilms, can form and these have been reported in the context of lung infections, otitis media, non-healing wounds and soft tissue fillers. It is unclear whether the same bacterial traits are required for aggregate formation as for biofilm formation. In this report, using isogenic mutants, we demonstrate that P. aeruginosa aggregates in liquid cultures are spontaneously formed independent of bacterial flagellar motility and independent of an exogenous scaffold. This contrasts with the role of the flagellum to initiate surface-adhered biofilms. Similar to surface attached biofilms, these aggregates exhibit increased antibiotic tolerance compared to planktonic cultures. These findings provide key insights into the requirements for aggregate formation that contrast with those for biofilm formation and that may have relevance for the persistence and dissemination of non-motile bacteria found within chronic clinical infections. IMPORTANCE: In this work, we have investigated the role of bacterial motility with regard to antibiotic-tolerant bacterial aggregate formation. Previous work has convincingly demonstrated that P. aeruginosa flagellar motility promotes the formation of surface-adhered biofilms in many systems, while, in contrast, aggregate formation was observed only by non-motile but not by motile P. aeruginosa in the presence of an exogenous scaffold. Here we identify that both wildtype P.aeruginosa and mutants that genetically lack motility spontaneously form antibiotic-tolerant aggregates in the absence of an exogenously-added scaffold. Additionally, we also demonstrate that wildtype and non-motile P. aeruginosa bacteria can co-aggregate, shedding light on potential physiological interactions and heterogeneity of aggregates.