Fig. 1: Experimental design of the antibiofilm evolution assay.

Experimental setup of the evolution assay to extend the host range and improve the antimicrobial and antibiofilm activity of bacteriophages (phages). a Biofilm formation on porous glass beads by incubation with P. aeruginosa in tryptic soy broth (TSB) under agitation and 37 °C. Dip-washing of beads with 24 h pre-established biofilm in sterile phosphate-buffered saline (PBS) before transferal into the calorimetric ampules. b Representation of one calorimetric ampule containing a 24 h pre-stablished biofilm bead of one P. aeruginosa strain in TSB and the phage mixture at round 0 (R0) containing the four ancestral phages. A total of 43 ampules (one growth control and four phage mixture dilutions per strain and three sterility controls) were used during each round of evolution. c Heat production (J) was recorded for each individual ampule by isothermal calorimetry during 24 h. d At the end of each round, the percentage heat reduction of phage-containing ampules relative to growth control ampules was determined at a 24 h (rounds 1–15) or 8 h (rounds 16–30) time point. Samples showing a heat reduction equal or above 75% (referred to as active samples) were selected and pooled together with the undiluted phage samples (always included) into the new phage mixture. e Across all eight bacterial strains, the undiluted and the active samples after each round were pooled together, creating the phage mixture that served as starting point for the next round, and hence was added to pre-stablished biofilm beads anew. In total, 30 rounds of evolution were performed. Figure 1 was created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licences/by-nc-nd/4.0/deed.en).