Comparative proteomic profiling in constructed tri-species consortia of Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae

Polymicrobial infections involving Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), and
Klebsiella pneumoniae (KP) are clinically significant, often leading to worsened outcomes, elevated
antibiotic resistance, and increased tolerance. A key challenge in studying long-term microbial
interactions is the propensity of PA to dominate in vitro cultures, limiting observations of co-adaptation.
To overcome this, we employed adaptive laboratory evolution over 15 growth cycles, exposing PA to
cell-free supernatant-conditioned media from SA and KP. The evolved strains (SASn-evolved and KPSnevolved) exhibited phenotypic shifts in biofilm formation, pyocyanin production, and antibiotic
susceptibility, while proteomic analysis revealed conserved alterations in pathways related to type VI
secretion, phenazine biosynthesis, β-lactam resistance, cytotoxicity, and growth. Whole-genome
sequencing identified the mutations in ampG, dipA, anmK, bifA, and rpoS among supernatant-evolved
strains.
To further investigate interspecies adaptation, we developed a tri-species co-culture system (PA, SA, KP)
supplemented with bovine serum albumin to prolong bacterial survival. After 6 days of co-culture, when
SA and KP populations showed significant reduction, we isolated SA and KP, followed by initiating a
cyclical maintenance strategy with 9-day intervals for both reintroduction of fresh SA and KP isolates and
complete medium replacement. After 36 days, PA isolates displayed heterogeneous adaptations—some
exhibited enhanced biofilm and pyocyanin production, while others showed impaired pyocyanin
biosynthesis capacity. Fitness assays demonstrated divergent evolutionary trajectories, with half of PA
isolates showing increased relative fitness, whereas others remained neutral or impaired. Comparative
proteomic profiling of pre- and post-evolution strains enabled quantification of 2943 protein groups on
average, providing insights into species-specific adaptations.
Current efforts focus on refining analytical frameworks for polymicrobial proteomics to elucidate better
the molecular mechanisms governing interspecies interactions.