The mechanism of chloramphenicol resistance was examined in a high-level-resistant isolate of Pseudomonas cepacia from a patient with cystic fibrosis. We investigated potential resistance mechanisms, including production of chloramphenicol acetyltransferase, ribosomal resistance, and decreased permeability. This strain (MIC, 200 ,ug/ml) had no detectable chloramphenicol acetyltransferase activity. In in vitro translation experiments in which we compared the resistant isolate with a susceptible strain of P. cepacia, inhibition of amino acid incorporation was equivalent even in organisms that were preincubated with sub-MICs of chloramphenicol. A 21.9-kilobase (kb) fragment of DNA was cloned which coded for chloramphenicol resistance; this fragment was expressed in P. cepacia but not in Escherichia coli. Quantitation of chloramphenicol uptake in the isogenic pair of susceptible and resistant organisms revealed a nearly 10-fold decrease of drug entry into the resistant strain. Comparison of isolated outer membrane proteins and lipopolysaccharide patterns identified no significant differences between the isogenic pair of organisms. We concluded that the mechanism of chloramphenicol resistance in this strain is decreased permeability.Pseudomonas cepacia is an important nosocomial pathogen and an organism that can cause severe pulmonary infections in children and young adults with cystic fibrosis (CF). Originally recognized as a plant pathogen in 1950 (5), the role of P. cepacia in hospital-acquired infections of the urine, lungs, and bloodstream was first reported in the early 1970s (12,19,22,28,35). Colonization with P. cepacia occurs late in the course of pulmonary disease in patients with CF, and in several CF centers, outbreaks of virulent strains have resulted in severe and overwhelming pneumonia in a subpopulation of patients (21, 37).The major problem in the treatment of infections caused by P. cepacia is multiple antibiotic resistance. Isolates from patients with CF, in particular, are often highly resistant to the P-lactam agents and aminoglycosides that are used to treat Pseudomonas aeruginosa (4, 9, 15). Antibiotics with clinical efficacy against P. cepacia include chloramphenicol, trimethoprim, and the quinolones (12,21,28). However, more than half of clinical isolates may be resistant to these agents as well. Investigations of chloramphenicol resistance in P. cepacia strains from patients with CF consistently report that 45 to 50% of isolates are resistant (MIC,.10 ,ug/ml).In this report we describe the characterization of chloramphenicol resistance in P. cepacia. We used an isolate of P. cepacia from a patient with CF; this isolate was resistant to chloramphenicol, trimethoprim, and ciprofloxacin, as well as all P-lactams and aminoglycosides tested, and was selected to characterize the resistance mechanism. We cloned a 21.9-kilobase (kb) DNA fragment that encoded resistance to chloramphenicol in P. cepacia but that was not expressed in Escherichia coli. Potential mechanisms of resistance, including enzy...