Stenotrophomonas maltophilia (Sm) is an opportunistic multi-resistant nosocomial pathogen which forms biofilms. Biofilms, microorganisms that grow on a surface within a matrix of extracellular polymeric substances (EPS), are highly resistant to antibiotics and host immune defenses. In Sm quorum sensing positively regulates biofilm formation and virulence. Sm produces two chromosomal inducible β- lactamases: L1, a molecular class B Zn2+-dependent metalloenzyme, and L2, an ampR-linked class A cephalosporinase. Previous studies in Sm K279a and K279a ampRFS (ampR deletion of 121 bp) showed that the induction of both β-lactamases depends on AmpR. AmpR regulates the expression of ampC in Enterobacteriaceae, while in Pseudomonas aeruginosa also regulates genes involved in biofilm production and virulence. The aim of this work was to assess the role of AmpR on S. maltophilia biofilm formation, EPS production and virulence. Studies were done on Sm K279a, and on two mutants obtained by Mathew Avison (University of Bristol, UK): K279a ampRFS and K279aM11 (constitutively active AmpR). Biofilm formation in microtitre plates was quantified by crystal violet staining and the  architecture of biofilms stained with Syto9 was analyzed by CLSM. K279a ampRFS produced higher amounts of biofilms than K279a (P < 0.01) and CLSM analysis demonstrated improved biofilm organization. On the other hand, K279aM11
produced lower amounts of biomass than K279a (P < 0.001). Confocal images acquired from K279a and K279aM11 biofilms showed a confluent growth with microcolonies scattered on the surface, while K279a ampRFS showed enhanced extent and complexity of biofilms with several big aggregates. In 3D reconstructions peaks of 30 μm in height were observed in K279a and K279aM11 biofilms, whereas K279a ampRFS showed taller peaks up to 70 μm. The EPS production, evaluated in microplates by calcofluor staining, was higher in K279a ampRFS biofilms (P < 0.001). K279a ampRFS also produced higher amounts of EPS than the other strains in planktonic cultures (ethanol precipitation), but the difference was not significant. All strains presented EPS with a similar composition, as revealed by ATR-FTIR spectroscopy. The virulence of S. maltophilia strains was evaluated by infecting larvae of Galleria mellonella. A statistically significant difference in G. mellonella killing was observed among the strains, being K279a ampRFS the most virulent (P < 0.01). The enhanced virulence could be related to the formation of biofilms with greater biomass and complexity and the production of higher amounts of EPS. These results suggest that AmpR negatively regulates S. maltophilia biofilm formation, EPS production and virulence. AmpR has been mentioned as a potential target for antimicrobials to overcome the β-lactam resistance of S. maltophilia; however, this strategy could be dangerous because of the role of AmpR on biofilm formation and virulence.