Antibiotic Resistance Protocols (Methods in Molecular Biology)

98

1. From a frozen stock, streak out wild-type E. coli MG1655 for
   single colonies on a Muller-Hinton agar plate. Incubate over-
   night at 37 °C (see Note 1).


2. Begin ten separate 2 mL liquid overnight cultures in Muller-
   Hinton media in 10 mL disposable Falcon tubes. Use a sepa-
   rate colony to inoculate each tube. Incubate the tubes for 24 h
   at 37 °C with vigorous aeration (200 rpm) (see Notes 2 and
   3 ).


3. 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 for
   subsequent analysis.


4. Transfer 2 μL of each culture into a new tube containing 2 mL
   of fresh Muller-Hinton media with 0.016 μg/mL ciprofloxa-
   cin (a concentration corresponding to the MIC of E. coli
   MG1655). Incubate the tubes at 37 °C for 24 h with vigorous
   aeration (see Notes 4 and 5 ).


5. Transfer 2 μL of each culture into a new tube containing 2 mL
   of fresh Muller-Hinton media with 0.024 μg/mL ciprofloxa-
   cin (a concentration corresponding to 1.5× the MIC of E. coli
   MG1655). Incubate the tubes at 37 °C for 24 h with vigorous
   aeration (see Notes 4 and 5 ).


6. 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 for
   subsequent analysis.


7. Repeat steps 5 and 6 , increasing the concentration of antibi-
   otic 1.5-fold at each cycle. Continue until the desired level of
   resistance is achieved.


8. Store the final culture as a frozen stock at − 80 °C for subse-
   quent analysis.

Whole genome sequence analysis is the method of choice to deter-

mine the mutational events that occurred during the selected evo-

lution of resistance to an antibiotic.

1. Cultures from the frozen stocks from the final serial transfer
   step (Subheading 3.1, step 8) are struck out for single colonies
   and incubated overnight at 37 °C on Mueller-Hinton II agar
   plates containing the same concentration of ciprofloxacin as
   was used in the final step of selection (see Note 6).


2. Single colonies are picked and used to inoculate independent
   tubes containing 2 mL of Mueller-Hinton II broth supple-
   mented again with the same concentration of ciprofloxacin.
   These tubes are incubated for 16–24 h at 37 °C with vigorous
   aeration (200 rpm).

3.2 Whole Genome
Sequencing

Douglas L. Huseby and Diarmaid Hughes