Download Degree project in Biology

The impact of different combinations of resistance-associated mutations on
fluoroquinolone resistance in Escherichia coli
Marja Rasinperä
Fluoroquinolones are antimicrobial drugs that prevent the replication of the bacterial
chromosome. Fluoroquinolones interact with their target molecules, DNA gyrase and
topoisomerase IV, enzymes that are essential in the replication. When a fluoroquinolone binds
toone of these enzymes it cannot function properly, with the result that the bacterial cell cannot
divide successfully and gradually dies. However, bacteria have found ways to avoid the effects of
these and other antibiotics, in becoming resistant. Resistance can be natural or acquired. Natural
resistance is a property typical for certain bacterial species. In acquired resistance a bacterial
strain that has been susceptible to the drug becomes resistant. Escherichia coli was originally
susceptible to fluoroquinolones and they have been used with success, for example, in the
treatment of urinary tract infections (UTI) that are mostly caused by E. coli. However, as a
consequence of the increased use of the drug some strains have developed resistance to
fluoroquinolones.
Many different mutations causing resistance to fluoroquinolones have been
observed in the drug target genes, and in genes that are associated with drug import and export
(efflux) in bacterial cells. Enhanced resistance due the presence of a plasmid-encoded targetprotection protein has also been observed. A plasmid is a self-replicating extra-chromosomal
circular DNA molecule. However, the development of a clinically relevant level of resistance
(greater than can be treated effectively) requires accumulation of multiple mutations in different
resistance-associated genes.
The purpose of this project was to construct strains carrying combinations of
mutations similar to those that have been observed in resistant strains of clinical UTI isolates. E.
coli strains with single mutations in either drug target genes or with deletion (loss) of import or
efflux-associated genes were available in the lab. From these, I constructed strains that each had
one of three different target gene mutations and one or two of six different deletions of import or
efflux-associated genes. After constructing the strains I determined the minimum inhibitory
concentrations (MIC) of ciprofloxacin, one of the important fluoroquinolone drugs. MIC is the
lowest concentration of the antibiotic that inhibits bacterial growth. MIC values were compared
to those of the wild type strain without any mutations in resistance-associated genes.
All the strains with target gene mutations resuted in some increase of the MIC
values. This was expected because most of the resistant strains observed in clinical isolates have
mutations in these genes. Deletions of drug import (porin) genes combined with target gene
mutations did not cause increased MIC values of the strains. Deletions in one or two effluxassociated genes acrR or acrR + marR had no effect on the MIC values although some increase
would have been expected because they repress the function of the efflux pump. However a
partial deletion in the acrR caused somewhat increased MIC values. When looking at these
results it can be seen that single mutations do not have much or any effect on the resistance and
the MIC values start to increase when more mutations are accumulated in the same strain.
Degree project in Biology, Examensarbete i biologi, 10 p autumn 2005
Biology Education Centre and Department of Cell and Molecular Biology, Uppsala University
Supervisor/Co-supervisor: Diarmaid Hughes/Linda Marcusson