Related Experiment Videos
Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon
1Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada.
Abstract:
An outer membrane protein of 50 kDa (OprK) was overproduced in a siderophore-deficient mutant of Pseudomonas aeruginosa capable of growth on iron-deficient minimal medium containing 2,2'-dipyridyl (0.5 mM). The expression of OprK in the mutant (strain K385) was associated with enhanced resistance to a number of antimicrobial agents, including ciprofloxacin, nalidixic acid, tetracycline, chloramphenicol, and streptonigrin. OprK was inducible in the parent strain by growth under severe iron limitation, as provided, for example, by the addition of dipyridyl or ZnSO4 to the growth medium. The gene encoding OprK (previously identified as ORFC) forms part of an operon composed of three genes (ORFABC) implicated in the secretion of the siderophore pyoverdine. Mutants defective in ORFA, ORFB, or ORFC exhibited enhanced susceptibility to tetracycline, chloramphenicol, ciprofloxacin, streptonigrin, and dipyridyl, consistent with a role for the ORFABC operon in multiple antibiotic resistance in P. aeruginosa. Sequence analysis of ORFC (oprK) revealed that its product is homologous to a class of outer membrane proteins involved in export. Similarly, the products of ORFA and ORFB exhibit homology to previously described bacterial export proteins located in the cytoplasmic membrane. These data suggest that ORFA-ORFB-oprK (ORFC)-dependent drug efflux contributes to multiple antibiotic resistance in P. aeruginosa. We propose, therefore, the designation mexAB (multiple efflux) for ORFAB.
Insights
The Pseudomonas aeruginosa ORFABC operon, including the outer membrane protein OprK (ORFC), contributes to multiple antibiotic resistance. This efflux system enhances bacterial defense against various antimicrobial agents.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Genetics
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen known for its intrinsic and acquired multidrug resistance.
- Outer membrane proteins play a crucial role in bacterial cell envelope structure and function, including antibiotic resistance.
- Siderophore-mediated iron uptake systems can influence bacterial physiology and drug resistance.
Purpose of the Study:
- To investigate the role of the outer membrane protein OprK (ORFC) and its associated operon (ORFABC) in the multidrug resistance of Pseudomonas aeruginosa.
- To determine if the ORFABC operon is involved in the efflux of antimicrobial agents.
- To characterize the genetic basis of enhanced antibiotic resistance observed in specific Pseudomonas aeruginosa mutants.
Main Methods:
- Overproduction of the 50 kDa outer membrane protein OprK in a siderophore-deficient Pseudomonas aeruginosa mutant.
- Growth of bacterial strains under iron-limited conditions using 2,2'-dipyridyl.
- Assessing antimicrobial resistance profiles of wild-type, mutant, and OprK-overproducing strains.
- Sequence analysis of the ORFABC operon and homology searches for its gene products.
Main Results:
- Overexpression of OprK in a mutant strain conferred enhanced resistance to ciprofloxacin, nalidixic acid, tetracycline, chloramphenicol, and streptonigrin.
- The ORFABC operon, involved in pyoverdine secretion, was found to be essential for multiple antibiotic resistance.
- Mutants lacking functional ORFA, ORFB, or ORFC genes showed increased susceptibility to various antibiotics and dipyridyl.
- Sequence analysis indicated that ORFABC gene products are homologous to bacterial export proteins.
Conclusions:
- The ORFABC operon, including OprK (ORFC), functions as a multidrug efflux system in Pseudomonas aeruginosa.
- This efflux mechanism contributes significantly to the bacterium's ability to resist multiple antimicrobial agents.
- The proposed designation mexAB (multiple efflux) for ORFAB is supported by these findings.