99- Transfer 900 μL of each overnight culture to 2 mL screw-cap
tubes containing 225 μL of sterile 50% glycerol in Muller-
Hinton II broth. These tubes should be stored at − 80 °C (see
Note 7). - The remaining culture should be used to prepare genomic
DNA. A wide range of commercial kits is available for the prep-
aration of genomic DNA. - The genomic DNA should be whole-genome sequenced.
There are many options for whole-genome sequencing. Most
groups have their own sequencing capabilities, use an institu-
tional core facility, or take advantage of one of a number of
commercial options (see Note 8). - The resultant sequence should be analyzed versus a reference
sequence for SNPs, deletions, insertions, and copy-number
variants. - It is likely that multiple mutations will have accumulated, rais-
ing at least two questions that will need to be addressed. (a)
What is the order in which individual mutations occurred? (b)
What is the nature of the contribution of individual mutations
to the selected phenotype? - Question (a), the order in which mutations accumulated, can
be addressed by repeating WGS steps 1– 6 on clones isolated
from the stocks made of earlier steps in the evolutionary serial
passage experiment. - Question (b) can be addressed by using genetics to reconstruct
isogenic strains carrying suitable combinations of mutations
identified from the WGS analysis and then measuring their
MIC (described in Subheading 3.3 below) and measure their
relative fitness under an appropriate condition [ 8 , 9 ]. - Relating MIC and relative fitness with the data from WGS is
expected to give a useful insight into the nature of the selective
advantage conferred by each successive mutation that accumu-
lated during the experimental evolution experiment.
The minimal inhibitory concentration (MIC) of an antibiotic is
defined as the lowest concentration of drug that, under a defined
set of agreed conditions, prevents visible growth of a bacterial cul-
ture. The procedure that follows outlines the conditions that
should be met and the procedures that should be followed when
using broth dilution, in microtiter plate format, to measure
MIC. The conditions follow closely those recommended by
EUCAST [ 10 ] and are applicable to E. coli and fluoroquinolones,
but details may need to be changed depending on the growth
requirements of particular bacterial species and the properties of
particular antibiotics being tested.3.3 MIC Assay
by Broth Dilution
Determining Trajectories of Bacterial Resistance Evolution