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resistant to one of the antibiotics used in the chromogenic agar
and susceptible to the antibiotic used in blood agar and anaer-
obic agar plates. We suggest that phenotypic and genotypic
characterization of isolates is performed prior to investigation,
to ensure easy and effective identification of isolate in feces,
e.g., MAST-test, MLST, and RAPD. RAPD is used for succes-
sive identification. For a similar study it would be possible to
use other ESBL- or carbapenemase-producing E. coli of other
ST’s or serogroups; such as B2-O16-ST16, ST69 or ST153.- Prior to investigation all plates must be tested. At least three
chromogenic agar plates from each batch are tested with one
susceptible and one resistant Enterobacteriaceae and finally one
vancomycin-susceptible Enterococcus spp. Store at 4–5 °C and
keep out of light. Alternatively cover them with tinfoil. - CFU counts of CP suspensions are performed as described for
the fecal samples to verify the inoculum. - A single daily subcutaneous dose can produce similar levels of
drugs in mice feces, to those seen in humans [ 31 ]. Saline solu-
tion containing antibiotics should be prepared on a daily basis
to ensure a precise and correct concentration. Information on
dilution and storage should be done according to instructions
from manufacturer and further guidance can be found from
current literature, such as Andrews et al. [ 47 ]. Solutions should
be kept in dark or covered bottles to avoid disintegration and
change in concentration. All antibiotics are dissolved in sterile
0.9% saline to avoid any pain or wounding of mice during
administration. - We include a control group receiving the CP but no antibiotic
treatment and one group receiving treatment with cefotaxime
with no inoculation with CP. - For this study we used ampicillin, cefotaxime, cefuroxime, cip-
rofloxacin, meropenem, dicloxacillin, mecillinam, clindamycin,
and metronidazole, but other antibiotics can be applied. - New sets of tweezers are used for each cage.
- Between each serial passage the pipette tip should be changed
and the solution vortexed. The plates used for the spot CFU
method should be completely dry. If this is not the case the
spots will mix with each other. Dry your plates beforehand in
an incubator. - Example of CFU calculations:
CFU spots are performed on duplicate plates using the
same dilution row. Colonies are therefore counted in two
spots for each dilution on each type of plate. The samples are
diluted tenfold (0.1 mL of sample diluted in 0.9 mL of 0.9%
saline). Thus, first spot is undiluted, the second is diluted ten-
Frederik Boëtius Hertz et al.