Bacterial infections often occur in polymicrobial biofilms where nutrient limitation and interspecies interactions can profoundly shape microbial physiology. Enterococcus faecalis can antagonize Pseudomonas aeruginosa growth under conditions of iron limitation, such as those found in the mammalian host. Here, we report that this growth antagonism reveals surviving P. aeruginosa cells capable of surviving antibiotic challenge, including ampicillin, cefepime, and ciprofloxacin, when grown in iron-restricted biofilms with E. faecalis . Transcriptomic profiling of P. aeruginosa revealed a distinctive response characterized by broad downregulation of biosynthetic, metabolic, and virulence pathways, alongside selective induction of membrane remodeling proteins, transport systems, and biofilm-associated genes. Induction of arnT in P. aeruginosa , required for lipid A modification, correlated with enhanced antibiotic survival to ampicillin, cefepime, and ciprofloxacin. Additionally, the diguanylate cyclase SiaD and efflux transporter MfsC in P. aeruginosa were implicated in decreased antibiotic susceptibility to the same antibiotics. This transcriptional response was unique to the dual stress of iron deprivation and microbial competition with E. faecalis , illustrating how interspecies interactions can simultaneously inhibit and protect P. aeruginosa , shedding light on potential persistence mechanisms in iron-limited polymicrobial environments.

IMPORTANCE : This study addresses antibiotic susceptibility in Pseudomonas aeruginosa , a major opportunistic ESKAPE pathogen, within polymicrobial biofilms and under host-relevant iron-restricted conditions. Polymicrobial biofilm-associated infections are notoriously difficult to treat due to complex interspecies interactions and increased antibiotic resilience. We demonstrate that Enterococcus faecalis not only antagonizes P. aeruginosa growth under iron limitation but also induces a unique transcriptional profile, enhancing P. aeruginosa survival during antibiotic challenge. This shift involves broad transcriptional reprogramming in P. aeruginosa , characterized by global metabolic downregulation and activation of envelope remodeling pathways, including the arn operon. These findings reveal how interspecies interactions under iron stress can both suppress and protect bacterial pathogens and underscore the importance of considering community context in treatment strategies for persistent infections.