The modulating effect of N-acetylcysteine (NAC) on the activity of different antibiotics has been studied in Pseudomonas aeruginosa. Our results demonstrate that, in contrast to previous reports, only the activity of imipenem is clearly affected by NAC. MIC and checkerboard determinations indicate that the NAC-based modulation of imipenem activity is dependent mainly on OprD. SDS-PAGE of outer membrane proteins (OMPs) after NAC treatments demonstrates that NAC does not modify the expression of OprD, suggesting that NAC competitively inhibits the uptake of imipenem through OprD. Similar effects on imipenem activity were obtained with P. aeruginosa clinical isolates. Our results indicate that imipenem-susceptible P. aeruginosa strains become resistant upon simultaneous treatment with NAC and imipenem. Moreover, the generality of the observed effects of NAC on antibiotic activity was assessed with two additional bacterial species, Escherichia coli and Acinetobacter baumannii. Caution should be taken during treatments, as the activity of imipenem may be modified by physiologically attainable concentrations of NAC, particularly during intravenous and nebulized regimes.A lthough its benefit has been questioned (1), N-acetylcysteine (NAC) is being used for the treatment of numerous disorders, including paracetamol intoxication, doxorubicin cardiotoxicity, ischemia-reperfusion cardiac injury, acute respiratory distress syndrome, bronchitis, chemotherapy-induced toxicity, HIV/ AIDS, heavy metal toxicity, and psychiatric disorders (2). This compound is also sold as a dietary supplement, with claims of antioxidant and liver-protecting effects. The antioxidant activity of NAC has been attributed to its reactivity with ·OH, ·NO2, CO 3 · Ϫ , and thiyl radicals, to its capacity for repair of oxidized key cellular molecules, and to its activity as a precursor for glutathione biosynthesis (2). Because of this mucolytic capacity, it has also been used to facilitate the processing of sputum specimens in bacteriology laboratories (3). The ability of NAC to reduce biofilms, alone or in combination with antimicrobials, has been addressed in several bacterial species (4-6). Furthermore, NAC has been proposed as a treatment in Helicobacter pylori infections (7). NAC utility in reducing sputum viscosity in patients with cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) (8), likely due to its ability to break disulfide bonds, has also been claimed, although its mechanism of action is not well understood.As a result of an imbalance between the production of reactive oxygen species (ROS) caused by inflammation and their inactivation by the impaired antioxidant systems, CF patients with chronic Pseudomonas aeruginosa lung infection have increased oxidative stress. Therefore, antioxidant interventions, including the use of NAC, have been proposed to reduce the extent of oxidative lesions and the rate of lung deterioration (for a review, see reference 9). However, although controversial results on the improvement of lung f...