During chronic infection, several factors contribute to the biogeography of microbial communities. Heterogeneous populations of Pseudomonas aeruginosa form aggregates in cystic fibrosis airways; however, the impact of this population heterogeneity on spatial organization and aggregate assembly is not well understood.

It is now well established that bacteria live in structured aggregates during chronic infections, where they evolve to adapt to the host environment in order to evade host immune responses and therapeutic interventions. Despite recent developments on how the physical properties of polymers impact on aggregate formation, changes in bacterial properties to overcome this have been overlooked. Here we show that even with physical entropic forces imposed by polymers in a sputum medium environment, lipopolysaccharide (LPS) plays a crucial role in aggregate assembly in Pseudomonas aeruginosa by altering the hydrophobicity of the cell surface. Our findings highlight that in chronic infections such as the polymer rich (eDNA and mucin) airways in cystic fibrosis (CF) lungs, O-antigen can dictate the type of aggregate assembly allowing the cells to overcome entropic forces, and sheds new light on the benefits or loss of O-antigen in polymer rich environments such as CF lungs.ImportanceDuring chronic infection, several factors contribute to the biogeography of microbial communities. Heterogeneous populations of Pseudomonas aeruginosa form aggregates in cystic fibrosis airways, however, the impact of this population heterogeneity on spatial organization and aggregate assembly is not well understood. In this study we found that changes in O-antigen structure determine the spatial organization of P. aeruginosa cells by altering the relative cell surface hydrophobicity.