The objective of this work was to investigate the risk factors for the acquisition of Pseudomonas aeruginosa and its resistance phenotypes in critically ill patients, taking into account colonization pressure. METHODS: We conducted a prospective cohort study in an 8-bed medical intensive care unit during a 35-month period. Nasopharyngeal and rectal swabs and respiratory secretions were obtained within 48 hours of admission and thrice weekly thereafter. During the study, a policy of consecutive mixing and cycling periods of three classes of antipseudomonal antibiotics was followed in the unit. RESULTS: Of 850 patients admitted for ≥ 3 days, 751 (88.3%) received an antibiotic, 562 of which (66.1%) were antipseudomonal antibiotics. A total of 68 patients (8%) carried P. aeruginosa upon admission, and among the remaining 782, 104 (13%) acquired at least one strain of P. aeruginosa during their stay. Multivariate analysis selected shock (odds ratio (OR) = 2.1; 95% confidence interval (CI), 1.2 to 3.7), intubation (OR = 3.6; 95% CI, 1.7 to 7.5), enteral nutrition (OR = 3.6; 95% CI, 1.8 to 7.6), parenteral nutrition (OR = 3.9; 95% CI, 1.6 to 9.6), tracheostomy (OR = 4.4; 95% CI, 2.3 to 8.3) and colonization pressure >0.43 (OR = 4; 95% CI, 1.2 to 5) as independently associated with the acquisition of P. aeruginosa, whereas exposure to fluoroquinolones for >3 days (OR = 0.4; 95% CI, 0.2 to 0.8) was protective. In the whole series, prior exposure to carbapenems was independently associated with carbapenem resistance, and prior amikacin use predicted piperacillin-tazobactam, fluoroquinolone and multiple-drug resistance. CONCLUSIONS: In critical care settings with a high rate of antibiotic use, colonization pressure and non-antibiotic exposures may be the crucial factors for P. aeruginosa acquisition, whereas fluoroquinolones may actually decrease its likelihood. For the acquisition of strains resistant to piperacillin-tazobactam, fluoroquinolones and multiple drugs, exposure to amikacin may be more relevant than previously recognized.