Strains were inoculated into 2mL of tryptic soy broth (TSB) from blood agar plates and incubated at 37°C for 6h. Cultures were then diluted 1:100 in TSB and TSB supplemented with 4% NaCl. 200μl of the suspension were then transferred to a 96-well cell culture plate (Nunclon Delta Surface, Thermo Fisher Scientific, Waltham, MD, USA) and incubated for 18-20h at 37°C under static growth conditions. OD600 was recorded and supernatant media was then removed and the remaining biomass gently washed three times with sterile PBS in order to remove non-adherent cells. 96-well plates were left to dry and then stained with 100μl of crystal violet per well for 10min. Wells were washed again and Biofilm formation was quantified by assessing the absorbance at 570 nm and 405 nm as a reference wavelength. Experiment were performed in duplicates with four technical replicates each.

The stability of the biofilm phenotype was tested on a subset of two isolates each from six infections and was maintained over 12 generations. Thus, the phenotype of each analysed clone is stable and heterogeneity is not due to stochastic fluctuations.

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