Fig. 1: Biofilms of clinical P. aeruginosa isolates fall into three major clusters independent of their phylogenetic background.

a Despite a large structural diversity in biofilms of 414 clinical isolates, groups of strains that share structural characteristics were identified by visual inspection of biofilm microscopy images. Biofilms were grown for 48 h in a microtiter plate-based in vitro biofilm assay; images were acquired using confocal laser scanning microscopy (CLSM) following live/dead staining. Living cells are displayed in green (Syto9); dead cells in red (propidum iodide: PI). 3D reconstructions were generated with the Imaris Software. The scale bar represents 50 µm. b Representative biofilm images of selected clinical isolates show exemplarily the structural characteristics of the three major biofilm clusters, of which each contains 59 strains (cluster A), 38 strains (cluster B), and 47 strains (cluster C), respectively. c The phylogenetic relationship of 33 representative clinical isolates is displayed in a phylogenetic tree based on 3524 genes that are present in the DNA sequences of 414 clinical isolates and 5 reference strains. The color code of the strain names represents the affiliation to a certain biofilm cluster: Red—cluster A; green—cluster B; blue—cluster C. Reference strains are displayed in black. The proportion of PAO1-like strains is highlighted in light gray; PA14-like strains are highlighted in dark gray. d Crystal violet quantification was performed for 33 representative clinical strains to assess air–liquid biofilm formation on a PVC surface after 24 h. Each datapoint represents one clinical isolate. Statistical significance was calculated using Tukey’s HSD (honest significant difference) following analysis of variance (ANOVA) and is displayed as *p < 0.05.