A permeability barrier as a mechanism of chloramphenicol resistance in Haemophilus influenzae.
Chloramphenicol resistance in Haemophilus influenzae occurs most frequently via plasmid-mediated chloramphenicol acetyltransferase production. We studied four strains with high-level chloramphenicol resistance (MIC greater than 20 micrograms/ml) which did not have detectable chloramphenicol acetyltransferase activity. The chloramphenicol resistance determinant was transformed into a chloramphenicol-susceptible laboratory H. influenzae strain from each of the four wild-type strains, enabling isogenic comparisons. By thin-layer chromatography and a bioassay, there was no evidence of non-chloramphenicol acetyltransferase modification of chloramphenicol. In vitro protein synthesis in the presence of chloramphenicol was equivalently inhibited in the chloramphenicol-resistant transformants and in the susceptible recipient. Chloramphenicol uptake by these strains during logarithmic growth was compared by high-pressure liquid chromatographic quantitation; at chloramphenicol concentrations of 5, 10, and 20 micrograms/ml the four transformants showed a decreased rate of uptake of chloramphenicol compared with the isogenic chloramphenicol-susceptible recipient. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of outer membrane proteins revealed a markedly diminished 40-kilodalton protein in the resistant transformants. We propose that the mechanism of chloramphenicol resistance in these strains is a relative permeability barrier due to the loss of an outer membrane protein.
D000123:Acetyltransferases / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis; D002463:Cell Membrane Permeability; D002701:Chloramphenicol / Q000378:metabolism / Q000494:pharmacology*; D015500:Chloramphenicol O-Acetyltransferase; D002855:Chromatography, Thin Layer; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000302:isolation & purification; D004352:Drug Resistance, Microbial; D004587:Electrophoresis, Agar Gel; D006193:Haemophilus influenzae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D009693:Nucleic Acid Hybridization; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D002701:Chloramphenicol; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase