Antibiotic tolerance due to restriction of cAMP-Crp regulation by mannitol, a non-glucose-family PTS carbon source

Enzyme-IIA (EIIA , Crr) of the phosphotransferase system (PTS) connects the uptake of glucose-family sugars to the cAMP-Crp regulatory cascade; phosphorylated EIIA enhances cAMP-Crp activity, which then contributes to the antibiotic-mediated accumulation of reactive oxygen species (ROS) and cell death. Defects in PTS cause antibiotic and disinfectant tolerance. We report that mannitol, a carbon source whose uptake does not use EIIA , reduces antibiotic-mediated killing of without affecting antibiotic minimal inhibitory concentration. Thus, mannitol promotes antibiotic tolerance. The tolerance pathway was defined by the loss of ciprofloxacin lethality from the deletion of (first gene in PTS), (mannitol-specific Enzyme-II), (cAMP synthase), and (cAMP receptor protein) but not (EIIA ). A mutant, which encodes a constitutively active Crp that bypasses the need for cAMP activation, also decreased mannitol-mediated antibiotic tolerance, as did exogenous cAMP. Thus, inhibition of antibiotic lethality by mannitol involves both PTS-mediated mannitol uptake and suppression of cAMP-Crp action, independent of EIIA . Mannitol suppressed the downstream antibiotic-mediated transcription of genes involved in NADH production and cellular respiration, expression of a superoxide reporter gene ( ), and accumulation of antibiotic-mediated ROS. Similar phenomena were observed with mannose and sorbitol, demonstrating that non-glucose PTS carbon sources can cause antibiotic tolerance by a novel path that reduces the ROS-promoting activity of cAMP-Crp. The work emphasizes that antibiotic tolerance, which contributes to disease relapse and the need for prolonged antibiotic treatment, can result from commonly consumed carbohydrates. This finding, plus mutations that interfere specifically with antibiotic lethality, makes tolerance a high probability event.IMPORTANCEBacterial tolerance constitutes a significant threat to anti-infective therapy and potentially to the use of disinfectants. Deficiency mutations that reduce glucose uptake, central carbon metabolism, and cellular respiration confer antibiotic/disinfectant tolerance by reducing the accumulation of reactive metabolites, such as reactive oxygen species. We identified novel environmental generators of tolerance by showing that non-glucose carbohydrates, such as mannitol, mannose, and sorbitol, generate tolerance to multiple antibiotic classes. Finding that these sugars inhibit a universal, stress-mediated death pathway emphas