Related Experiment Videos
Resistance to trimethoprim in Haemophilus influenzae
M Powell1, Y Hu, D M Livermore
1London Medical College, UK.
Abstract:
The mechanisms of resistance to trimethoprim in eleven U.K. clinical isolates of Haemophilus influenzae were studied. The levels of dihydrofolate reductase (DHFR) activities in crude extracts from four resistant wild-types were similar to those in susceptible controls. However, activities in extracts from the other seven resistant wild-type isolates, and transformants of two of these, were at least triple those in the sensitive strains. Resistance to trimethoprim was also selected for in vitro during prolonged exposure to the drug and was associated with increased levels of DHFR specific activity in the mutants. DHFR enzymes were, however, still very susceptible to inhibition by trimethoprim. Activities in four extracts, including one from a transformant of a resistant mutant, were reduced by at least 45% following incubation with 10(-8) M trimethoprim. The results suggested that overproduction of the chromosomal DHFR enzyme may be the resistance mechanism in some organisms. The much lower DHFR activities measured in extracts from other resistant isolates may reflect synthesis of chromosomal enzymes that have reduced susceptibility to trimethoprim.
Insights
Mechanisms of trimethoprim resistance in Haemophilus influenzae involve dihydrofolate reductase (DHFR). Some isolates show increased DHFR activity, while others may have altered DHFR enzymes less susceptible to trimethoprim.
Area of Science:
- Microbiology
- Molecular Biology
- Pharmacology
Background:
- Trimethoprim is a crucial antibiotic targeting dihydrofolate reductase (DHFR).
- Understanding trimethoprim resistance mechanisms in Haemophilus influenzae is vital for effective treatment.
- Clinical isolates of H. influenzae can develop resistance to trimethoprim through various pathways.
Purpose of the Study:
- To investigate the mechanisms of trimethoprim resistance in eleven U.K. clinical isolates of Haemophilus influenzae.
- To determine the role of dihydrofolate reductase (DHFR) activity and susceptibility in trimethoprim resistance.
- To explore potential genetic alterations leading to trimethoprim resistance.
Main Methods:
- Analysis of dihydrofolate reductase (DHFR) enzyme activity in crude extracts from resistant and susceptible H. influenzae isolates.
- In vitro selection of trimethoprim resistance through prolonged drug exposure.
- Transformation experiments to confirm the genetic basis of resistance.
- Assessment of DHFR enzyme susceptibility to trimethoprim inhibition.
Main Results:
- Four resistant isolates showed DHFR activity levels similar to susceptible controls.
- Seven resistant isolates exhibited DHFR activities at least triple those of sensitive strains.
- In vitro selected mutants displayed increased DHFR specific activity.
- DHFR enzymes from resistant isolates remained highly susceptible to trimethoprim inhibition (e.g., 45% reduction at 10(-8) M).
Conclusions:
- Overproduction of chromosomal DHFR enzyme is a likely resistance mechanism in some H. influenzae isolates.
- Altered chromosomal DHFR enzymes with reduced susceptibility to trimethoprim may also contribute to resistance.
- Further research is needed to fully elucidate the genetic and enzymatic basis of trimethoprim resistance in H. influenzae.