Enterococcus faecalis alters antibiotic susceptibility in Pseudomonas aeruginosa mixed species biofilms

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, a known host defense mechanism. We report here that this growth antagonism uncovers 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 above. 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.