THE PSEUDOMONAS AERUGINOSA BIOFILM INDUCTION RESPONSE TO SUBINHIBITORY ANTIBIOTICS REQUIRES oprF AND sigX

Abstract

Pseudomonas aeruginosa is a Gram-negative pathogen that forms biofilms, which increase tolerance to antibiotics. Biofilms are dense, surfaceassociated communities of bacteria that grow in a self-produced matrix of polysaccharides, proteins, and extracellular DNA (eDNA). Sub-minimal inhibitory concentration (sub-MIC) levels of antibiotics induce the formation of biofilms, indicating a potential role in response to antibiotic stress. However, the mechanisms behind sub-MIC antibiotic-induced biofilm formation are unknown. We show that treatment with sub-MIC levels of cefixime (cephalosporin), carbenicillin (β-lactam), tobramycin (aminoglycoside), chloramphenicol (chloramphenicol), thiostrepton (thiopeptide), novobiocin (aminocoumarin), ciprofloxacin (fluoroquinolone), or trimethoprim (antifolate) induces biofilm formation, with maximal induction at ~ ¼ to ½ MIC. We demonstrate that addition of exogenous eDNA or cell lysate does not stimulate biofilm formation to the same extent as antibiotics, suggesting that the release of common goods by antibiotic action does not solely drive the biofilm response. We show that increased biofilm formation upon antibiotic exposure requires the outer membrane porin OprF and the extracytoplasmic function sigma factor SigX. Through transposon mutant screening and deletion studies, we found that OprF is important for biofilm induction, as mutants lacking this protein did not form increased biofilm when exposed to sub-MIC antibiotics. OprF expression is v controlled by SigX, and its loss increases SigX activity. Loss of SigX also prevents biofilm induction by sub-MIC antibiotics. Together, these results show that antibiotic-induced biofilm formation may constitute a type of stress response. This response may be useful to screen for new antibiotics due to its ability to reveal antibiotic activity at concentrations below the MIC. Further study of this response may also provide targets for adjuvant therapies that reduce biofilm formation in P. aeruginosa infections and increase the efficacy of current antibiotics.