Anoxia selects for high fitness biofilms and increased antibiotic resistance in Pseudomonas aeruginosa
Antibiotic resistance is a growing global health crisis, yet resistance is almost exclusively quantified under aerobic laboratory conditions that fail to reflect the complex microenvironments bacteria encounter during infection. Many clinically important infection sites, such as airways of individuals with cystic fibrosis or chronic wounds, are microaerobic to anoxic. To address this, we investigated how anoxia alters antibiotic resistance determinants, hypothesizing that anaerobic metabolism might change the fitness effects and selection of resistance mutations. We used experimental evolution to propagate Pseudomonas aeruginosa populations for approximately 200 generations under conditions differing in oxygen availability (oxic vs. anoxic), growth mode (biofilm vs. planktonic), and tobramycin (TOB) exposure (subinhibitory increasing to inhibitory concentrations). Subinhibitory exposure was sufficient to achieve resistance 2-4x greater than ancestral levels, with anoxic populations consistently having higher minimum inhibitory concentrations than oxic populations. While resistance mutations in fusA1 and ptsP arose across all conditions, mutations in amgS were only selected in oxic populations - indicating condition-specific resistance mutations. Notably, mexT mutations were observed in anoxic or tobramycin-exposed populations. The presumed inactivation of mexT may also enhance virulence through altered quorum sensing and increased rhamnolipid production. Anoxic populations additionally exhibited significantly increased biofilm formation, reduced twitching motility driven by type IV pilus gene mutations, and greater competitive fitness. Together, these findings demonstrate that oxygen availability shapes resistance evolution in P. aeruginosa, with the anoxic environment selecting for a more virulent, sessile, and antibiotic-resistant phenotype. ImportancePseudomonas aeruginosa is an opportunistic pathogen capable of acute and chronic infections that develop antibiotic resistance. One aspect of P. aeruginosa that makes it so challenging to treat is its metabolic versatility. We wanted to understand how growth in different infection-relevant conditions, specifically anoxia and surface attachment, would alter the evolutionary pathways of antibiotic resistance. We found that the mutations causing resistance to the commonly used antibiotic tobramycin were condition dependent. We also observed that adaptation to anoxia resulted in P. aeruginosa populations that had high biofilm forming capacity and were highly fit compared to its ancestor. This indicates that anoxic infection environments can lead to P. aeruginosa variants with increased resistance, increased recalcitrance, and potentially more virulence.