Experimental evolution reveals adaptive pathways to reduced antibiotic susceptibility in Pseudomonas aeruginosa biofilms.

In the present study, we used an experimental evolution approach to investigate how reduced antibiotic susceptibility develops in biofilms formed by Pseudomonas aeruginosa when they are exposed to the clinically relevant antibiotics tobramycin and meropenem. Two biofilm model systems were used, one mimicking surface-attached biofilms (multiple independent P. aeruginosa lineages originating from P. aeruginosa AA2-1 and maintained for 4-10 cycles) and one mimicking biofilm aggregates in cystic fibrosis sputum (multiple independent P. aeruginosa lineages originating from 6 different reference strains and maintained for 15 cycles). Whole-genome sequencing of evolved populations revealed both known and previously unknown genetic adaptations that reduce antibiotic susceptibility. While reduced susceptibility to meropenem commonly arose from mutations in oprD, we also identified a role for irlR. The development of reduced susceptibility towards tobramycin was more complex, with mutations in fusA1, anr, ccoQ1, ptsP, phoQ and nppA1A2BCD associated with adaptation to this antibiotic. Our findings underscore the multifaceted strategies employed by P. aeruginosa to withstand antibiotic pressure in biofilms.