Low levels of the second messenger c-di-GMP enhance tolerance and resistance to meropenem in Pseudomonas aeruginosa

Background: The carbapenem antibiotic meropenem is often used to treat lifethreatening infections caused by Pseudomonas aeruginosa. Previous studies have shown that the susceptibility of P. aeruginosa to carbapenems is differentially regulated by the RNA chaperone Hfq, depending on the availability of preferred or less preferred carbon sources, a mechanism known as carbon catabolite repression (CCR). In this regulation, Hfq plays a CCR-conditioned repressive role on outer membrane porins that act as entry ports for carbapenems. In this study, we investigated whether meropenem response is modulated by the second messenger c-di-GMP, which is known to regulate several bacterial functions. Methods: We used P. aeruginosa strains with high or low c-di-GMP levels and their Hfq-deficient derivatives to assess the role of c-di-GMP in modulating meropenem susceptibility and tolerance. Results: We show that low intracellular c−di−GMP levels increase meropenem resistance and tolerance at sub−inhibitory concentrations, whereas high c−di −GMP diminishes both traits. Importantly, c−di−GMP status shapes the entire response trajectory, from exponential growth to the stationary phase. Furthermore, we show that c-di-GMP modulates meropenem response through mechanism(s) independent of Hfq-mediated porin repression and exerts a dominant effect over CCR-driven regulation. Conclusion: This study supports the notion that P. aeruginosa meropenem susceptibility and tolerance are modulated by intracellular c-di-GMP concentrations, with low c-di-GMP levels promoting higher fitness. Our findings indicate that c-di-GMP exerts its regulatory effect through mechanisms distinct from Hfq-mediated porin control, underscoring the existence of parallel regulatory pathways that shape antibiotic response.