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Chloramphenicol resistance in Pseudomonas cepacia because of decreased permeability
J L Burns1, L A Hedin, D M Lien
1Division of Infectious Disease, Children's Hospital and Medical Center, Seattle, Washington 98105.
Abstract:
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 micrograms/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.
Insights
This study investigated chloramphenicol resistance in Pseudomonas cepacia. The high-level resistance was found to be due to decreased drug permeability, not enzyme activity.
Area of Science:
- Microbiology
- Molecular Biology
- Pharmacology
Background:
- Pseudomonas cepacia is an opportunistic pathogen often found in cystic fibrosis patients.
- Antibiotic resistance in P. cepacia poses a significant clinical challenge.
- Understanding resistance mechanisms is crucial for effective treatment strategies.
Purpose of the Study:
- To elucidate the mechanism of high-level chloramphenicol resistance in a P. cepacia isolate.
- To investigate potential roles of chloramphenicol acetyltransferase, ribosomal resistance, and decreased permeability.
Main Methods:
- MIC determination for chloramphenicol.
- In vitro translation assays comparing resistant and susceptible strains.
- DNA fragment cloning and expression studies.
- Quantification of chloramphenicol uptake.
- Outer membrane protein and lipopolysaccharide analysis.
Main Results:
- The P. cepacia isolate exhibited high-level chloramphenicol resistance (MIC = 200 µg/ml).
- No chloramphenicol acetyltransferase activity was detected.
- In vitro translation inhibition was similar between resistant and susceptible strains.
- A 21.9 kb DNA fragment conferring resistance was cloned and expressed in P. cepacia, but not E. coli.
- Resistant strain showed a nearly 10-fold decrease in chloramphenicol uptake.
- No significant differences in outer membrane proteins or LPS were observed.
Conclusions:
- Decreased permeability is the primary mechanism of chloramphenicol resistance in this P. cepacia strain.
- The resistance determinant appears specific to P. cepacia.
- This finding highlights the importance of drug influx in bacterial antibiotic resistance.