Elevated levels of the second messenger c-di-GMP contribute to antimicrobial resistance of Pseudomonas aeruginosa

Biofilms are highly structured, surface-associated communities. A hallmark of biofilms is their extraordinary resistance to antimicrobial agents that is activated during early biofilm development of Pseudomonas aeruginosa and requires the regulatory hybrid SagS and BrlR, a member of the MerR family of multidrug efflux pump activators. However, little is known about the mechanism by which SagS contributes to BrlR activation or drug resistance. Here, we demonstrate that sagS biofilm cells harbour the secondary messenger c-di-GMP at reduced levels similar to those observed in wild-type cells grown planktonically rather than as biofilms. Restoring c-di-GMP levels to wild-type biofilm-like levels restored brlR expression, DNA binding by BrlR, and recalcitrance to killing by antimicrobial agents of sagS biofilm cells. We likewise found that increasing c-di-GMP levels present in planktonic cells to biofilm-like levels (55pmol mg(-1)) resulted in planktonic cells being significantly more resistant to antimicrobial agents, with increased resistance correlating with increased brlR, mexA, and mexE expression and BrlR production. In contrast, reducing cellular c-di-GMP levels of biofilm cells to 40pmol mg(-1) correlated with increased susceptibility and reduced brlR expression. Our findings suggest that a signalling pathway involving a specific c-di-GMP pool regulated by SagS contributes to the resistance of P.aeruginosa biofilms.