Highly effective modulator therapy hinders the emergence of hyperbiofilm variants of <i>Pseudomonas aeruginosa</i> PA14 grown in a cystic fibrosis lung model

<jats:title>Abstract</jats:title> <jats:p> <jats:italic>Pseudomonas aeruginosa</jats:italic> is an opportunistic pathogen that often adopts persistent phenotypes — such as biofilm formation— that are associated with chronic infections including those observed in the cystic fibrosis (CF) lung Recently, highly effective modulator therapy (HEMT) such as elexacaftor/tezacaftor/ivacaftor (ETI) has significantly improved the quality of life of people with CF (pwCF). Yet a potential direct impact of ETI on the physiology of <jats:italic>P. aeruginosa</jats:italic> during growth to a remodeled CF lung environment has remained unexplored. To address this, we conducted an experimental evolution using <jats:italic>P. aeruginosa</jats:italic> PA14 grown in CF-like conditions in the presence or absence of ETI. We observed a marked reduction in biofilm formation and in the number of small colony variants (SCVs) for <jats:italic>P. aeruginosa</jats:italic> populations evolved under ETI treatment. Also, sequencing of specific evolved clones exhibiting distinct morphotypes revealed two major observations: (i) <jats:italic>P. aeruginosa</jats:italic> -evolved communities exposed to ETI retained a wild type-like morphotype and, (ii) <jats:italic>P. aeruginosa</jats:italic> populations evolved in the absence of ETI adopted a SCV-like phenotype with mutations acquired in the Wsp chemosensory pathway. Furthermore, analysis of evolved populations revealed that ETI treatment likely modulates c-di-GMP pools by driving mutations in an enzyme catalyzing the degradation of this second messenger. Overall, our work suggests that ETI has the potential to hinder the acute to chronic biofilm transition of <jats:italic>P. aeruginosa</jats:italic> thereby limiting the emergence of variants typically associated with long-term CF lung colonization. </jats:p>