Antibiotic Resistance Protocols (Methods in Molecular Biology)

97

for culture and drug dilutions throughout the assay. Make up

according to the manufacturer’s instructions.

2. Mueller-Hinton II agar: (from Becton Dickinson, cat. no.
   211441) for bacterial growth. Make up according to the man-
   ufacturer’s instructions.


3. It is not possible to give details here (there are too many details
   and different options available) other than to state that there
   are several commercially available kits for the preparation of
   genomic DNA, and for its preparation into libraries suitable
   for whole genome sequencing. Currently popular sequencing
   technologies are marketed under the Illumina and PacBio
   brands, but there are others. Many users will probably make
   use of in house genome sequencing facilities, or commercial
   companies that perform all of the steps and also provide bioin-
   formatics analysis services.


4. Mueller-Hinton II broth (MHII): cation-adjusted (Becton
   Dickinson, cat. no. 212322) for bacterial growth, and for cul-
   ture and drug dilutions throughout the assay. Make up accord-
   ing to the manufacturer’s instructions.


5. 96-well (12 × 8) microtiter plates: use round-bottomed plates
   (VWR, cat. no. 732-2725).


6. Sterile polyester adhesive film for microplates (VWR, cat. no.
   60941-064).


7. Microwell lid for 96-well plates (VWR, cat. no. 734-2185).

3 Methods

The goal of serial transfer experiments is to recreate selections that

bacteria may face that lead to the development of antibiotic

resistance. To this end, the choice of selective conditions is abso-

lutely essential. Below is described a liquid-media selection in

which the concentration of the fluoroquinolone antibiotic cipro-

floxacin is increased in 1.5-fold steps. Practically, the described

experiment leads to the development of E. coli mutants that are

clinically resistant to ciprofloxacin (EUCAST breakpoint of 1 μg/

mL) after 14 cycles.

Depending on the experimental question being addressed, dif-

ferent selection regimes will be called for. Liquid serial transfer experi-

ments, similar to the one described below, impose a selection pressure

for fast growth in addition to antibiotic resistance. Similar experi-

ments done by plating for single colonies on solid media containing

antibiotics will, in contrast, select for resistance almost exclusively.

2.2 Whole Genome
Sequencing
Components

2.3 Minimal
Inhibitory
Concentration (MIC)
Components

3.1 Experimental
Evolution by Serial
Transfer

Determining Trajectories of Bacterial Resistance Evolution