Download Relationship between mutation and resistance to fluoroquinolones

Relationship between mutation and resistance to fluoroquinolones in
Escherichia coli.
Julia Paschke
Bacteria develop resistance against the important group of antibiotics, the fluoroquinolones, in
a multistep process by accumulating different kinds of mutations in several genes. The
antibiotics fluoroquinolones are used to treat urinary tract infections, which are caused in 80%
of the cases by the bacterium Escherichia coli. They affect essential activities of bacterial
enzymes that are important for DNA multiplication and cell division. If the enzymes are
blocked by the small fluoroquinolones, cell metabolism and division is inhibited and the E.
coli bacteria expire. When these enzymes are altered through mutation in the gene gyrA,
resistance of bacteria against fluoroquinolones can increase. Also alterations of an efflux
pump system, which pumps drugs out of the cell, can increase the resistance of bacteria
against fluoroquinolones. The altered efflux pump system exports drugs more rapidly out of
the cell, and therefore the fluoroquinolones can not harm the bacteria. Other resistanceassociated mutations are in genes for proteins called porins in the outer membrane that are
crucial for import into the cell. Mutations affecting porins inhibit the entry of the
fluoroquinolones into the cell.
The aim of this project was to investigate a correlation of those different mutations to the
resistance level of the bacteria. Different E.coli strains had been constructed in the way that
they carry different resistance-associated mutations (by Linda Marcusson, unpublished data).
Each of three strains carried a different mutation in the gyrA gene. Other resistance-associated
mutations had been introduced into these three strains to construct strains with multiple
combinations of mutations, like they are found in some clinical isolates. This was done
through phage transduction, which means the transfer of host genes from one cell to another
by a virus. Sequencing, the determination of the DNA sequence, was performed to identify
the altered genes in the right place of the bacterial genomes.
The different strains containing diffeent combinations of resistance mutations were examined
through measuring the Minimum Inhibitory Concentrations (MICs) for diffeent antibiotics.
This is a standard way to determine the resistance level of bacterial strains. The Minimum
Inhibitory Concentration corresponds to the resistance level of the bacteria. The results
showed that one of the gyrA mutations had a much higher effect on the resistance level than
the other two gyrA mutations. The efflux pump mutations enhanced the resistance levels of
the gyrA mutants. These mutations combined with the mutations of the outer membrane
porins gave highest levels of resistance. Thus, as expected, the combination of all three
classes of mutations in one bacterial strain had a major affect on the resistance level.
Interestingly, one of the efflux pump mutations appeared to have no effect on the resistance
level and counteracted the effect of a different mutation of the efflux pump system.
Degree project in Biology, summer 2005
Examensarbete i biologi, 10p, sommar 2005
Department of Biology Education and
Department of Cell and Molecular Biology, Microbiology
Supervisor: Prof. Diarmaid Hughes and PhD student Linda L. Marcusson