Pseudomonas cepacia is emerging as an important pulmonary pathogen both in children and young adults with cystic fibrosis and in hospitalized patients who acquire pneumonia. Prevention of acquisition of P. cepacia is difficult because it is ubiquitous in the hospital environment and therapy is severely limited by resistance of the organism to multiple antimicrobial agents. The most clinically effective antibiotics include chloramphenicol and trimethoprim/sulfamethoxazole. A survey of the antimicrobial susceptibility of clinical isolates of P. cepacia revealed chloramphenicol resistant strains which do not have detectable chloramphenical acetyltransferase activity. The two mechanisms of chloramphenicol resistance that have been reported in Gram-negative organisms are production of chloramphenicol acetyltransferase and limited drug permeability. Thus, the hypothesized resistance mechanism in these strains is decreased chloramphenicol entry, offering a unique opportunity to investigate membrane transport in P. cepacia. The proposed research will seek to confirm that chloramphenicol resistance is due to a permeability barrier and to characterize the nature of that barrier. The approach will be to mobilize the resistance gene by molecular cloning to enable comparison of strains that differ only in their susceptibility to chloramphenicol. Invetigation of other potential resistance mechanisms in isogenic susceptible and resistant strains will include assays for enzymatic inactivation of chloramphenicol and quantitation of ribosomal sensitivity to inhibition by chloramphenicol. Although decreased drug penetration will be assumed if no other resistance mechanisms are identified, cellular uptake of chloramphenicol will be quantitated by high performance liquid chromatography. Utilizing a susceptible strain, chloramphenicol transport into both reconstituted outer membrane vesicles and cytoplasmic membrane vesicles will be characterized in P. cepacia. The mechanism of uptake and site of a permeability barrier will then be examined in a clone expressing resistance due to impermeability. Further understanding of the resistance mechanisms and physiology of P. cepacia will improve our ability to treat infections and control this emerging pathogen.
