Resumo

Following the genomic era, a large number of genes coding for enzymes predicted to synthesize and degrade 3'-5'-cyclic diguanylic acid (c-di-GMP) was found in most bacterial genomes and this nucleotide emerged as an important intracellular signal molecule controlling bacterial behavior. An association between high levels of c-di-GMP and antibiotic resistance is often assumed, since c-di-GMP regulates biofilm formation and this mode of growth leads to enhanced antibiotic resistance; however, a clear understanding of this correlation is missing. Here, through two-dimensional electrophoresis gels, we found that overexpression of c-di-GMP in Pseudomonas aeruginosa leads to decreased levels of five outer membrane porins, including OprD, responsible for the antibiotic imipenem uptake. We demonstrated that the strain with high c-di-GMP levels is sensitive to the same minimal inhibitory concentration (MIC) of imipenem as the wild type strain, but it showed a fitness advantage when grown at sub-MIC imipenem. Microscopic analysis using CFP-tagged strains demonstrated that the presence of imipenem provides a competitive advantage to cells with higher c-di-GMP over wild-type or low c-di-GMP levels strains. To rule out the effect of biofilm formation, we constructed a strain with high c-di-GMP levels in a pelD mutant background, that does not form biofilm and observed that this strain still present a fitness advantage in sub-MIC imipenem. The resistance mechanism is not due to transcriptional regulation of the β-lactamase or main efflux pumps genes, as analyzed by qRT-PCR. Our findings show that c-di-GMP levels modulate the fitness of P. aeruginosa in the presence of drugs through the modulation of outer membrane proteins and irrespective of the biofilm mode of growth; thus, regulation of c-di-GMP levels could represent a novel control approach against harmful bacteria.